Structural basis of species-specific endotoxin sensing by innate immune receptor TLR4/MD-2

Lipopolysaccharide (LPS), also known as endotoxin, activates the innate immune response through toll-like receptor 4 (TLR4) and its coreceptor, MD-2. MD-2 has a unique hydrophobic cavity that directly binds to lipid A, the active center of LPS. Tetraacylated lipid IVa, a synthetic lipid A precursor, acts as a weak agonist to mouse TLR4/MD-2, but as an antagonist to human TLR4/MD-2. However, it remains unclear as to how LPS and lipid IVa show agonistic or antagonistic activities in a species-specific manner. The present study reports the crystal structures of mouse TLR4/MD-2/LPS and TLR4/MD-2/lipid IVa complexes at 2.5 and 2.7 Å resolutions, respectively. Mouse TLR4/MD-2/LPS exhibited an agonistic “m”-shaped 2:2:2 complex similar to the human TLR4/MD-2/LPS complex. Mouse TLR4/MD-2/lipid IVa complex also showed an agonistic structural feature, exhibiting architecture similar to the 2:2:2 complex. Remarkably, lipid IVa in the mouse TLR4/MD-2 complex occupied nearly the same space as LPS, although lipid IVa lacked the two acyl chains. Human MD-2 binds lipid IVa in an antagonistic manner completely differently from the way mouse MD-2 does. Together, the results provide structural evidence of the agonistic property of lipid IVa on mouse TLR4/MD-2 and deepen understanding of the ligand binding and dimerization mechanism by the structurally diverse LPS variants.Toll-like receptors (TLRs) recognize and respond to diverse pathogenic components of microorganisms and provide the first line of defense against microbial infection (1, 2). Among the microbial components, endotoxic lipopolysaccharide (LPS) from a membrane component of Gram-negative bacteria elicits the potent innate immune response through the receptor complex of TLR4 and MD-2 (3, 4). Excessive exposure to LPS often causes exaggerated signaling via TLR4 and fatal septic shock (5, 6), which is associated with a high mortality (20–30%) and is the most common cause of death in intensive care units (5, 6).The lipid A moiety of LPS, which anchors LPS to the outer membrane of Gram-negative bacteria, is responsible for the immunostimulatory activity of LPS (7, 8). Lipid A consists of a 1,4′-bis-phosphorylated diglucosamine backbone to which variable lengths and numbers of acyl chains are covalently linked (8). The two phosphate groups are also important for the agonistic activity of lipid A because deletion of either phosphate group reduces the endotoxic activity (9, 10).TLR4 is a type I transmembrane protein composed of 22 extracellular leucine-rich repeats (LRRs), a transmembrane domain, and the Toll/IL-1 receptor domain (TIR domain) that is essential for TLR signaling and conserved among members of the Toll receptor family (1). TLR4 alone does not directly bind LPS and requires the coreceptor MD-2 (11). MD-2 is associated with the extracellular domain of TLR4 and is indispensable for LPS recognition (4). A member of the MD-2–related lipid-recognition protein family (12), MD-2 directly binds to LPS in its hydrophobic cavity with high affinity (13).Recently, the crystal structure of human TLR4/MD-2/Ra-LPS (Ra chemotype of Escherichia coli LPS) complex (14) was solved, which revealed that five of the six acyl chains of LPS are buried inside the MD-2 cavity. The sixth acyl chain lies on the surface of MD-2, partially exposed to the solvent. Together with the hydrophobic residues of MD-2, the partially exposed acyl chain constitutes the secondary binding site for the hydrophobic patch on the C-terminal convex face of the horseshoe structure of TLR4, leading to the formation of the “m”-shaped 2:2:2 hTLR4/MD-2/LPS complex. The close proximity of the C terminus of the extracellular domain in the complex induced by binding to LPS may allow for dimerization and signaling by the intracellular TIR domains (15, 16).The number and length of the acyl chains determine the agonistic property of lipid A (17–19). E. coli lipid A is usually hexaacylated and acts as a potent agonist for all mammalian cells. In contrast, tetraacylated lipid IVa, the precursor of E. coli LPS, acts as an agonist only for some mammalian species. In particular, it acts as a weak agonist on mouse and as an antagonist on human cells (20, 21). Although several studies have investigated the species-specific activity of lipid IVa (22–28), these studies primarily used mutational and computational simulation methods. Structural information on the agonistic form of TLR4/MD-2 is limited to the hTLR4/MD-2/LPS complex; no structures of mTLR4/MD-2 complexed with LPS or lipid IVa are currently available. Structural knowledge may provide critical clues regarding the agonistic and antagonistic mechanisms by LPS and lipid IVa ligands that underlie species specificity.Here, we present the two agonistic structures of mouse TLR4/MD-2/Re-LPS (Re chemotype of E. coli LPS) and TLR4/MD-2/lipid IVa complexes at 2.5 and 2.7 Å resolutions, respectively. This structural study will provide better understanding of the LPS recognition and signaling mechanism and will contribute to the development of therapeutic antiseptic shock drugs targeting TLR4/MD-2.     

UNC93B1 is essential for the plasma membrane localization and signaling of Toll-like receptor 5

The proper trafficking and localization of Toll-like receptors (TLRs) are important for specific ligand recognition and efficient signal transduction. The TLRs sensing bacterial membrane components are expressed on the cell surface and recruit signaling adaptors to the plasma membrane upon stimulation. On the contrary, the nucleotide-sensing TLRs are mostly found inside cells and signal from the endolysosomes in an acidic pH-dependent manner. Trafficking of the nucleotide-sensing TLRs from the endoplasmic reticulum to the endolysosomes strictly depends on UNC93B1, and their signaling is completely abolished in the 3d mutant mice bearing the H412R mutation of UNC93B1. In contrast, UNC93B1 was considered to have no role for the cell surface-localized TLRs and signaling via TLR1, TLR2, TLR4, and TLR6 is normal in the 3d mice. Unexpectedly, we discovered that TLR5, a cell surface receptor for bacterial protein flagellin, also requires UNC93B1 for plasma membrane localization and signaling. TLR5 physically interacts with UNC93B1, and the cells from the 3d or UNC93B1-deficient mice not only lack TLR5 at the plasma membrane but also fail to secret cytokines and to up-regulate costimulatory molecules upon flagellin stimulation, demonstrating the essential role of UNC93B1 in TLR5 signaling. Our study reveals that the role of UNC93B1 is not limited to the TLRs signaling from the endolysosomes and compels the further probing of the mechanisms underlying the UNC93B1-assisted differential targeting of TLRs.Toll-like receptors (TLRs) sense unique microbial structures or host-derived molecules released from stressed or dying cells to initiate the innate immune responses (1). TLRs are composed of three domains: the leucine-rich repeat (LRR) domain responsible for ligand binding, a single transmembrane domain, and the cytoplasmic Toll/IL-1 receptor homology domain by which TLRs recruit adaptor molecules for downstream signal transduction. Activated TLRs stimulate the NF-κB, MAPK, and IFN regulatory factor pathways, leading to the expression of diverse inflammatory cytokines, chemokines, and type I interferons. TLRs also activate antigen presenting cells to induce costimulatory molecules and coordinate various aspects of adaptive immune responses (2).The members of the TLR family can be classified into two groups based on their subcellular localization patterns (3–5). TLR1, TLR2, TLR4, and TLR6, which mainly recognize the components of bacterial cell membrane, are located on the cell surface and initiate signaling thereat. In contrast, the nucleotide-sensing TLRs such as TLR3, TLR7, TLR8, TLR9, and TLR13 are largely found in endolysosomes and require an acidic environment for their efficient signaling. Additionally, TLR11 and TLR12, the sensors for Toxoplasma protein profilin, are also expressed inside cells and transmit signals in an acidic pH-dependent manner (6–8). All the intracellular TLRs commonly bind to a multispanning membrane protein UNC93B1, which is required for their proper localization and signaling (6–13). One missense mutation (H412R) of UNC93B1, found in a chemically mutagenized mouse strain called 3d, hinders binding of UNC93B1 with TLRs and prevents their exit from the endoplasmic reticulum (ER) (9–11). Consequently, signaling by all endosomal TLRs is abolished in the cells from 3d mice. In contrast, trafficking and signaling of the cell surface-localized TLRs such as TLR2 and TLR4 are not affected by the UNC93B1 mutation (9, 11).The proper localization of TLRs is critical not only for efficient signaling but also for preventing undesirable receptor hyperactivation (14, 15). Especially, sequestration of the nucleotide-sensing TLRs in endolysosomes significantly contributes to attenuating the immune stimulation by host-derived nucleotides abundant in the extracellular spaces (14). Structural discrimination of microbial vs. mammalian nucleotides is not straightforward, and a mutant TLR9 protein, engineered to artificially localize at the plasma membrane, responds to mammalian DNA as well as the CpG oligonucleotides mimicking bacterial DNA. As a result, mice expressing such mutant TLR9 succumb to systemic autoinflammation and die prematurely (15). Therefore, regulatory mechanisms for localization and trafficking of TLRs need to be tightly controlled.TLR5 recognizes flagellin, the major protein subunit of bacterial flagellum, and functions as a critical innate sensor for flagellated bacteria in all mucous organs (16–18). TLR5 plays an important role in intestinal homeostasis mediating the immune adaptation to symbiotic microflora as well as defense against pathogenic bacterial infection (19–21). In addition, systemic injection of flagellin confers protection against ionizing radiation in a TLR5-dependent manner, implying that TLR5 agonism might be clinically used for radioprotection (22). TLR5 overexpressed in the intestinal epithelial cells was exclusively found on the basolateral surface, accounting for the selective induction of proinflammatory cytokine by basolateral but not by apical flagellin (17). Also, we recently demonstrated that endogenous TLR5 is expressed at the cell surface of mouse neutrophils, monocytes, and dendritic cells (DCs) in a TLR-specific chaperone PRAT4A-dependnet manner (23). However, other regulatory mechanisms for the localization of TLR5 at the plasma membrane are unknown. Here, we show that UNC93B1 binds to TLR5, travels to the plasma membrane with the receptor, and is required for flagellin-induced signaling at the cell surface.     

Toll-like receptor 9 and 21 have different ligand recognition profiles and cooperatively mediate activity of CpG-oligodeoxynucleotides in zebrafish

CpG-oligodeoxynucleotides (CpG-ODNs) are potent immune stimuli currently under investigation as antimicrobial agents for different species. Toll-like receptor (TLR) 9 and TLR21 are the cellular receptors of CpG-ODN in mammals and chickens, respectively. The avian genomes lack TLR9, whereas mammalian genomes lack TLR21. Although fish contain both of these genes, the biological functions of fish TLR9 and TLR21 have not been investigated previously. In this study, we comparatively investigated zebrafish TLR9 (zebTLR9) and TLR21 (zebTLR21). The two TLRs have similar expression profiles in zebrafish. They are expressed during early development stages and are preferentially expressed in innate immune function-related organs in adult fish. Results from cell-based activation assays indicate that these two zebrafish TLRs are functional, responding to CpG-ODN but not to other TLR ligands. zebTLR9 broadly recognized CpG-ODN with different CpG motifs, but CpG-ODN with GACGTT or AACGTT had better activity to this TLR. In contrast, zebTLR21 responded preferentially to CpG-ODN with GTCGTT motifs. The distinctive ligand recognition profiles of these two TLRs were determined by their ectodomains. Activation of these two TLRs by CpG-ODN occurred inside the cells and was modulated by UNC93B1. The biological functions of these two TLRs were further investigated. The CpG-ODNs that activate both zebTLR9 and zebTLR21 were more potent than others that activate only zebTLR9 in the activation of cytokine productions and were more bactericidal in zebrafish. These results suggest that zebTLR9 and zebTLR21 cooperatively mediate the antimicrobial activities of CpG-ODN. Overall, this study provides a molecular basis for the activities of CpG-ODN in fish.Bacterial and viral CpG-deoxynucleotides containing DNA (CpG-DNA) represent a type of pathogen-associated molecular pattern (PAMP) that activates immune cells and triggers host responses to microbial infections (1–3). Synthetic phosphorothioate-modified CpG-oligodeoxynucleotides (CpG-ODNs) mimic the functions of CpG-DNA and have been investigated as immune modulators for their adjuvant and antimicrobial activities in different species (4–7). In general, a CpG-ODN contains one or more copies of CpG-deoxynucleotides containing hexamer motifs (CpG motifs). A CpG-ODN’s immunostimulatory activities are dependent on its length, the number of CpG motifs, and the position, spacing, and surrounding bases of these CpG motifs.A CpG-ODN can have varying immunostimulatory activity in different species. This species-specific property is determined by the nucleotide context of the CpG motifs within the CpG-ODN. For example, CpG-ODNs containing a purine-purine-CG-pyrimidine-pyrimidine motif, such as a GACGTT motif, are more potent in activating murine cells compared with those containing a GTCGTT motif. In contrast, the GTCGTT motif containing CpG-ODN generates stronger immune responses in humans and various domestic animals (8, 9).Toll-like receptors (TLRs) are pattern recognition receptors that play crucial roles in the initiation of host defense against microbial invasion by binding to PAMPs from the invading microorganisms. Ten TLRs (TLR1–TLR10) have been identified in human cells, and 13 have been identified in mouse cells. These TLRs detect diverse structures of PAMP from lipids, lipoproteins, glycans, and proteins to nucleic acids (10, 11). Of these, TLR9, a member of a subfamily of intracellular TLRs comprising TLR3, TLR7, TLR8, and TLR9, is the cellular receptor that mediates the functions of CpG-ODN. The species-specific activity of a CpG-ODN is attributed to a species-specific ligand recognition of TLR9 (12–14). In mammals, cellular localization and activation of TLR9 are regulated by various accessory proteins, including UNC93 Caenorhabditis elegans homolog of B1 (UNC93B1) (15–17). Activation of TLR9 by CpG-ODN results in various immunologic effects, including up-regulation of MHC class I and II costimulatory molecules, activation of natural killer cells and B cells, and increased B-cell proliferation. In addition, TLR9 activation up-regulates T helper (Th) 1-polarized cytokine production, which promotes T-cell activation. Because of these potent immunostimulatory effects, CpG-ODNs are currently under investigation for various therapeutic applications, including antitumor and anti-infection therapies and as vaccine adjuvants (18–20).Similar to their actions in mammalian species, in chickens CpG-ODNs activate marked immune responses and provide protection from microbial infections (4, 5, 21). Nevertheless, analysis of the chicken and zebra finch genomes found that the TLR9 gene is not present in avian genomes. Of the 10 avian TLRs, TLR1La, TLR1Lb, TLR2a, TLR2b, TLR3, TLR4, TLR5, and TLR7 are orthologs to mammalian TLRs, whereas TLR15 and TLR21 are not found in mammals (22). It was recently demonstrated that chicken TLR21 (chTLR21) is a functional homolog to mammalian TLR9 in terms of response to CpG-ODN stimulation (23, 24).The immunostimulatory effects of CpG-ODNs have been investigated in numerous fish species as well. In these species, much like in mammalian and avian species, CpG-ODNs up-regulate the activation of macrophages, induce proliferation of leukocytes, and stimulate cytokine expression. In addition, CpG-ODNs have been shown to protect fish against bacterial and viral infections. The molecular bases for CpG-ODN activation in fish remain unclear, however (5, 6). The genomic DNA of zebrafish has been sequenced and annotated, leading to the discovery of at least 14 different types of TLR in fish, including TLR9 and TLR21 (25, 26); however, whether these two TLRs are functional has not been investigated previously. In the present study, we comparatively investigated the expression, structural relationship, CpG-ODN interaction, regulation by UNC93B1, and immunologic functions of zebrafish TLR9 (zebTLR9) and TLR21 (zebTLR21) to explore the molecular basis of the immunostimulatory activities of CpG-ODN in fish.     

TLR-dependent phagosome tubulation in dendritic cells promotes phagosome cross-talk to optimize MHC-II antigen presentation

Dendritic cells (DCs) phagocytose large particles like bacteria at sites of infection and progressively degrade them within maturing phagosomes. Phagosomes in DCs are also signaling platforms for pattern recognition receptors, such as Toll-like receptors (TLRs), and sites for assembly of cargo-derived peptides with major histocompatibility complex class II (MHC-II) molecules. Although TLR signaling from phagosomes stimulates presentation of phagocytosed antigens, the mechanisms underlying this enhancement and the cell surface delivery of MHC-II–peptide complexes from phagosomes are not known. We show that in DCs, maturing phagosomes extend numerous long tubules several hours after phagocytosis. Tubule formation requires an intact microtubule and actin cytoskeleton and MyD88-dependent phagosomal TLR signaling, but not phagolysosome formation or extensive proteolysis. In contrast to the tubules that emerge from endolysosomes after uptake of soluble ligands and TLR stimulation, the late-onset phagosomal tubules are not essential for delivery of phagosome-derived MHC-II–peptide complexes to the plasma membrane. Rather, tubulation promotes MHC-II presentation by enabling maximal cargo transfer among phagosomes that bear a TLR signature. Our data show that phagosomal tubules in DCs are functionally distinct from those that emerge from lysosomes and are unique adaptations of the phagocytic machinery that facilitate cargo exchange and antigen presentation among TLR-signaling phagosomes.Professional phagocytes take up large particles, such as bacteria, by phagocytosis and submit them to an increasingly harsh environment during phagosome maturation (1). Phagocytes concomitantly alert the immune system that an invader is present via signaling programs initiated by pattern recognition receptors, such as Toll-like receptors (TLRs) (2). Conventional dendritic cells (DCs) also alter and optimize phagosome maturation and TLR-signaling programs to preserve bacterial antigens for loading onto MHC class I and class II (MHC-II) molecules and optimize cytokine secretion to stimulate and direct T-cell responses to the invading agent (3, 4). DC presentation of soluble antigen is facilitated by TLR-driven tubulation of lysosomes that harbor MHC-II–peptide complexes and by consequent fusion of tubulovesicular structures with the plasma membrane (5–7); however, little is known about the mechanism by which signaling pathways influence the formation or presentation of phagosome-derived MHC-II–peptide complexes, key processes in the adaptive immunity to bacterial pathogens.TLRs respond to microbial ligands at the plasma membrane and in intracellular stores (8). TLR stimulation at the plasma membrane, endosomes, or phagosomes elicits distinct signaling pathways via two sets of adaptors, TIRAP (or MAL)-MyD88 and TRAM-TRIF (8, 9), which induce proinflammatory cytokine secretion and other downstream responses. TLRs such as TLR2 and TLR4 are recruited to macrophage and DC phagosomes at least partly from an intracellular pool (10–13), and signal autonomously from phagosomes independent of plasma membrane TLRs (11, 14, 15). Autonomous phagosomal signaling from TLRs or Fcγ receptors enhances the degradation of phagocytosed proteins and assembly of MHC-II with their derived peptides (14–16). Phagosomal TLR signaling has been proposed to also promote the reorganization of phagosome-derived MHC-II-enriched compartments (MIICs) to favor the delivery of MHC-II–peptide complexes to the plasma membrane (17), analogous to TLR-stimulated formation of tubules from MIICs/lysosomes (18–20) that fuse with the plasma membrane (7) and extend toward the immunologic synapse with T cells (5). Tubules emerge from phagosomes in macrophages shortly after phagocytosis and likely function in membrane recycling during early phagosome maturation stages (21–23), but tubules at later stages that might facilitate the presentation of phagosome-derived MHC-II–peptide complexes have not been reported previously. Moreover, a role for TLR signaling in formation of phagosome-derived tubules has not been established.Herein we show that in DCs, maturing phagosomes undergo extensive tubulation up to several hours after phagocytosis, and that tubulation requires TLR and MyD88 signaling and an intact actin and microtubule cytoskeleton. Unlike lysosome tubulation, phagosome tubulation is not essential for MHC-II–peptide transport to the cell surface. Rather, it contributes to content exchange among phagosomes that carry a TLR signature, and thereby enhances presentation of phagocytosed antigens from potential pathogens.     

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein–protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C₁₆H₁₅NO₄ (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors.Toll-like receptors (TLRs) are type I transmembrane receptors that detect conserved “pathogen-associated molecular patterns” from microbes, as well as host-derived “danger-associated molecular patterns” (1). TLR2 heterodimerizes with TLR6 or TLR1 to recognize diacyl lipopeptides or triacyl lipopeptides, respectively (2, 3), present in gram-positive and gram-negative bacteria (4–9).Ligand engagement of TLR2/1 or TLR2/6 activates the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway (i.e., nuclear translocation of NF-κB, activation of MAPKs), resulting in production of proinflammatory cytokines (10). Dysregulated TLR2 signaling has been implicated in numerous diseases (e.g., sepsis, atherosclerosis, tumor metastasis, ischemia/reperfusion injury) (11–14). Several inhibitors of TLR2 signaling have been developed (15–18), yet none is licensed for human use. A better understanding of the Toll/IL-1 receptor resistance (TIR) domain interactions involved in TLR2 signaling could lead to novel therapeutic agents.Both TLRs and adapter proteins contain a cytoplasmic TIR domain that mediates homotypic and heterotypic interactions during TLR signaling (19). Two adapter proteins implicated in TLR2 signaling are MyD88 and TIRAP (Mal). A conserved Pro [e.g., P681 in human TLR2 (hTLR2), P712 in murine TLR4 (mTLR4), P674 in hTLR10, P804 in mTLR11] within the BB loop of almost all TIR domains is critical for signaling (20–27). More importantly, the BB loop P681H mutation in hTLR2 abolished recruitment of MyD88 and signaling (20, 26). Based on this evidence, the BB loop within the TLR2 TIR domain appears to be an ideal target for attenuation of TLR2 signaling.Visual inspection of the crystal structure of the hTLR2 TIR domain (26) revealed a pocket formed by residues on the β-B strand and α-B helix that includes the highly conserved Pro and Gly residues of the BB loop. We hypothesized that targeting this pocket with a small molecule might inhibit interaction of TLR2 with MyD88, and thereby blunt TLR2 signaling. We identified C₁₆H₁₅NO₄ (C29) and its derivative, ortho-vanillin (o-vanillin), which inhibit mTLR2 and hTLR2 signaling initiated by synthetic and bacterial agonists without cytotoxicity. Interestingly, mutation of the BB loop pocket residues revealed a differential requirement for TLR2/1 vs. TLR2/6 signaling. Our data indicate that computer-aided drug design (CADD) is an effective approach for identifying small molecule inhibitors of TLR2 signaling and has the potential to identify inhibitors for other TLR signaling pathways.     

TLR8 on dendritic cells and TLR9 on B cells restrain TLR7-mediated spontaneous autoimmunity in C57BL/6 mice

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with diverse clinical presentations characterized by the presence of autoantibodies to nuclear components. Toll-like receptor (TLR)7, TLR8, and TLR9 sense microbial or endogenous nucleic acids and are implicated in the development of SLE. In mice TLR7-deficiency ameliorates SLE, but TLR8- or TLR9-deficiency exacerbates the disease because of increased TLR7 response. Thus, both TLR8 and TLR9 control TLR7 function, but whether TLR8 and TLR9 act in parallel or in series in the same or different cell types in controlling TLR7-mediated lupus remains unknown. Here, we reveal that double TLR8/9-deficient (TLR8/9^−/−) mice on the C57BL/6 background showed increased abnormalities characteristic of SLE, including splenomegaly, autoantibody production, frequencies of marginal zone and B1 B cells, and renal pathology compared with single TLR8^−/− or TLR9^−/− mice. On the cellular level, TLR8^−/− and TLR8/9^−/− dendritic cells were hyperesponsive to TLR7 ligand R848, but TLR9^−/− cells responded normally. Moreover, B cells from TLR9^−/− and TLR8/9^−/− mice were hyperesponsive to R848, but TLR8^−/− B cells were not. These results reveal that TLR8 and TLR9 have an additive effect on controlling TLR7 function and TLR7-mediated lupus; however, they act on different cell types. TLR8 controls TLR7 function on dendritic cells, and TLR9 restrains TLR7 response on B cells.Systemic lupus erythematosus (SLE) is a complex chronic autoimmune disease that arises spontaneously and is characterized by production of autoantibodies against self-nucleic acids and associated proteins (1). These autoantibodies bind self-nucleic acids released by dying cells and form immune complexes that accumulate in different parts of the body, leading to inflammation and tissue damage. The kidneys, skin, joints, lungs, serous membranes, as well as, the cardiovascular, nervous and musculoskeletal system become targets of inflammation at onset or during the course of the disease (2). The etiology of SLE is unknown, yet genetics, sex, infectious agents, environmental factors, and certain medications may play a role in the initiation of the disease by causing alterations in lymphoid signaling, antigen presentation, apoptosis, and clearance of immune complexes (3, 4).Toll-like receptors (TLRs) detect specific microbial components widely expressed by bacteria, fungi, protozoa, and viruses, and initiate signaling pathways critical for induction of immune responses to infection (5). In contrast to the cell surface TLRs that detect bacterial cell wall components and viral particles, nucleic acid-sensing TLRs are localized mainly within endosomal compartments (6). Human endosomal TLRs consist of TLR3, which senses viral double-stranded RNA (dsRNA) (7), TLR7 and TLR8, which recognize viral single-stranded RNA (8–10), and TLR9, which detects bacterial and viral unmethylated CpG-containing DNA motifs (11). Interestingly, these endosomal TLRs are also able to detect self-nucleic acids (12–14). Although the endosomal localization isolate TLR3, TLR7, TLR8, and TLR9 away from self-nucleic acids in the extracellular space, still self-RNA or -DNA can become a potent trigger of cell activation when transported into TLR-containing endosomes, and such recognition can result in sterile inflammation and autoimmunity, including SLE (4, 15, 16). The connection of the endosomal TLRs with SLE originates mainly from mouse models, where TLR7 signaling seems to play a central role. TLR7 gene duplication is the cause for the development of lupus in mice bearing the Y chromosome-linked autoimmune accelerating (Yaa) locus that harbors 17 genes, including TLR7 (17, 18). In TLR7 transgenic mouse lines, a modest increase in TLR7 expression promotes autoreactive lymphocytes with RNA specificities and myeloid cell proliferation, but a substantial increase in TLR7 expression causes fatal acute inflammatory pathology and profound dendritic cell (DC) dysregulation (17). In addition, studies in several lupus-prone mouse strains have revealed that TLR7-deficiency ameliorates disease, but TLR9-deficiency exacerbates it. Interestingly, this controversy can be explained by the enhanced TLR7 activity in the TLR9-deficient lupus mice (19, 20). Although murine TLR8 does not seem so far to be able to sense a ligand (21, 22), we have shown previously that it plays an important biological role in controlling TLR7-mediated lupus. Indeed, TLR8-deficiency in mice (on the C57BL/6 background that is not prone to lupus) leads to lupus development because of increased TLR7 expression and signaling in DCs (23). Thus, tight control and regulation of TLR7 is pivotal for avoiding SLE and inflammatory pathology in mice. Recent studies in humans have also revealed that increased expression of TLR7 is associated with increased risk for SLE (24–26).Nucleic acid TLRs are expressed in many cell types, including DCs, plasmacytoid DCs (pDCs) and B cells, all of which play a central role in SLE development. TLR7, TLR8, and TLR9 signal through the adaptor molecule myeloid differentiation primary response gene 88 (MyD88), whereas TLR3 signals via the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor inducing IFN-β) (5). MyD88-deficiency abrogates most attributes of lupus in several lupus-prone mouse strains (19, 27–29). Moreover, deficiency for Unc93B1, a multipass transmembrane protein that controls trafficking of TLRs from the endoplasmic reticulum to endolysosomes and is required for nucleic acid-sensing TLR function (30), also abrogates many clinical parameters of disease in mouse lupus strains, suggesting that endosomal TLRs are critical in this disease (31). Interestingly, TLR9 competes with TLR7 for Unc93B1-dependent trafficking and predominates over TLR7 (32). TLR9 predominance is reversed to TLR7 by a D34A mutation in Unc93B1 and mice that carry this mutation show TLR7-dependent, systemic lethal inflammation (32).Thus, in mice both TLR8 and TLR9 control TLR7-mediated lupus, but it is unknown if these TLRs act in parallel or in series in the same or different cell types and if they have an additive effect or not in controlling TLR7. To address these issues, we generated double TLR8/TLR9-deficient (TLR8/9^−/−) mice and analyzed and compared the lupus phenotype in TLR8^−/−, TLR9^−/−, and TLR8/9^−/− mice. Our data revealed that TLR8/9^−/− mice have increased abnormalities characteristic of SLE and that both TLR8 and TLR9 keep TLR7-mediated lupus under control, but they act in different cell types. On DCs TLR7 function is ruled by TLR8, whereas on B cells TLR7 is mastered by TLR9.     

Essential requirement for IRF8 and SLC15A4 implicates plasmacytoid dendritic cells in the pathogenesis of lupus

In vitro evidence suggests that plasmacytoid dendritic cells (pDCs) are intimately involved in the pathogenesis of lupus. However, it remains to be determined whether these cells are required in vivo for disease development, and whether their contribution is restricted to hyperproduction of type I IFNs. To address these issues, we created lupus-predisposed mice lacking the IFN regulatory factor 8 (IRF8) or carrying a mutation that impairs the peptide/histidine transporter solute carrier family 15, member 4 (SLC15A4). IRF8-deficient NZB mice, lacking pDCs, showed almost complete absence of anti-nuclear, anti-chromatin, and anti-erythrocyte autoantibodies, along with reduced kidney disease. These effects were observed despite normal B-cell responses to Toll-like receptor (TLR) 7 and TLR9 stimuli and intact humoral responses to conventional T-dependent and -independent antigens. Moreover, Slc15a4 mutant C57BL/6-Fas^lpr mice, in which pDCs are present but unable to produce type I IFNs in response to endosomal TLR ligands, also showed an absence of autoantibodies, reduced lymphadenopathy and splenomegaly, and extended survival. Taken together, our results demonstrate that pDCs and the production of type I IFNs by these cells are critical contributors to the pathogenesis of lupus-like autoimmunity in these models. Thus, IRF8 and SLC15A4 may provide important targets for therapeutic intervention in human lupus.Extensive evidence suggests that type I IFNs are major pathogenic effectors in lupus-associated systemic autoimmunity. A well-documented pattern of expression of type I IFN-inducible genes occurs in peripheral blood mononuclear cells of patients with systemic lupus erythematosus (SLE) (1–3), and reduced disease is observed in some lupus-predisposed mice that either lack the common receptor (IFNAR) for these cytokines (4, 5) or have been treated with IFNAR-blocking antibody (6). Consequently, attention has focused on defining the cell subsets and signaling processes involved in type I IFN production, the mechanisms by which these mediators accelerate disease, and approaches to interfere with these pathogenic events.Early in vitro studies showed that type I IFN production can be induced in normal blood leukocytes by SLE autoantibodies complexed with nucleic acid-containing apoptotic/necrotic cell material, and further work demonstrated that this activity is sensitive to RNase and DNase digestion (7, 8). These results were integrated in a more comprehensive scheme following the demonstration that type I IFN induction by these complexes is mediated by the engagement of endosomal Toll-like receptors (TLRs) (9–11). Similarly, antigenic cargo containing nucleic acids was found to promote B-cell proliferation in a TLR9- or TLR7-dependent manner, with this effect enhanced by type I IFN signaling (9, 12, 13). The contribution of nucleic acid-sensing TLRs to systemic autoimmunity was further corroborated by studies in lupus-predisposed mice lacking or overexpressing TLR7 and/or TLR9 (14-20), and in Unc93b1 (3d) mutant mice in which signaling by endosomal TLRs is extinguished (21).The cell population involved in type I IFN production in response to lupus-related immune complexes corresponds to natural IFN-producing cells (22, 23). These cells, known as plasmacytoid DCs (pDCs), are the most potent producers of type I IFNs, a functional characteristic attributed to constitutive expression of TLR7, TLR9, and IRF7 and likely signaling from a unique intracellular compartment (24–27). The involvement of pDCs in lupus is further suggested by the reduced frequency of these cells in patient blood together with increases in afflicted organs, presumably caused by the attraction of activated pDCs to inflammatory sites (10). Similar increases have been noted in inflammatory tissues of patients with Sjögren''s syndrome (28), rheumatoid arthritis (29, 30), dermatomyositis (31), and psoriasis (32).Collectively, these results suggest that pDCs, acting through type I IFN hyperproduction, are major pathogenic contributors to lupus. Whether the participation of these cells is obligatory remains to be documented in vivo, however. Here, using congenic lupus-predisposed mice lacking pDCs (as well as other DC subsets) owing to IRF8 deficiency, or exhibiting pDC-specific defects in endosomal TLR signaling and type I IFN production owing to Slc15a4 (feeble) mutation, we provide strong evidence that pDCs are indeed required for disease development, and this effect appears to be mediated by hyperproduction of inflammatory cytokines, most likely type I IFNs.     

Autoinhibition and relief mechanism by the proteolytic processing of Toll-like receptor 8

Toll-like receptor 8 (TLR8) senses single-stranded RNA (ssRNA) and initiates innate immune responses. TLR8 requires proteolytic cleavage at the loop region (Z-loop) between leucine-rich repeat (LRR) 14 and LRR15 for its activation. However, the molecular basis of Z-loop processing remains unknown. To elucidate the mechanism of Z-loop processing, we performed biochemical and structural studies of how the Z-loop affects the function of TLR8. TLR8 with the uncleaved Z-loop is unable to form a dimer, which is essential for activation, irrespective of the presence of agonistic ligands. Crystallographic analysis revealed that the uncleaved Z-loop located on the ascending lateral face prevents the approach of the dimerization partner by steric hindrance. This autoinhibition mechanism of dimerization by the Z-loop might be occurring in the proteins of the same subfamily, TLR7 and TLR9.Toll-like receptors (TLRs) constitute a family of innate immune receptors that recognize pathogen-associated molecular patterns (1). The TLR molecule is a type I transmembrane protein characterized by an extracellular leucine-rich repeat (LRR) domain, a transmembrane helix, and an intracellular Toll/interleukin-1 receptor (TIR) homology domain (2). The typical TLR molecule is considered to be monomeric in the absence of ligands, transforming into an activated dimer form on ligand binding, which allows for dimerization of the intracellular TIR domain and subsequent signaling (2).The TLR subfamily comprising TLR7, TLR8, and TLR9 recognizes single-stranded (ss) nucleic acids from viruses and bacteria (3). Specifically, TLR7 and TLR8 recognize uridine- and guanosine-rich single-stranded RNA (ssRNA) (4–11), whereas TLR9 recognizes ssDNA containing the unmethylated cytosine-phosphate-guanine (CpG) dideoxynucleotide motif (12). Furthermore, TLR7 and TLR8 are also activated by synthetic chemical compounds (13, 14), such as imiquimod (TLR7-specific), resiquimod (R848; both TLR7 and TLR8), and CL075 (both TLR7 and TLR8).Certain regulation mechanisms of the functions of the TLR7–9 subfamily members are shared because of a high degree of sequence similarities (3). They reside on the endosomal membrane, and their transportation from endoplasmic reticulum (ER) to endolysosomes is mediated by the ER membrane protein Unc93B1 (15). Moreover, TLR7–9 possess a long inserted loop region (Z-loop), consisting of ∼30 amino acid residues, between LRR14 and LRR15, and the processing by proteolytic cleavage at the Z-loop is believed to be indispensable for their function (16–21). Specifically, the processing at the Z-loop of human TLR8 mediated by furin-like proprotein convertase and cathepsins produces functional TLR8 capable of ligand binding and signaling in endolysosomes. In addition, the cleaved form of TLR8 has been found to be predominant in immune cells (16). Recent structural studies demonstrate that the N- and C-terminal halves of TLR8 after Z-loop cleavage associate with each other, and that both fragments are cooperatively involved in ligand binding (22). Moreover, a recent study revealed that the latter half of the cleaved Z-loop interacts with LRRs to stabilize the TLR8 structure and contributes to ssRNA recognition by TLR8 (23).Although accumulating evidence illustrates the functional importance of Z-loop processing at the cellular level, mechanistic insights into this processing in the regulation of TLR8 function at the molecular level are lacking. Here, to unveil the mechanistic role of Z-loop processing of TLR8, we present the results of a combined structural and biochemical investigation of TLR8 with the uncleaved Z-loop.     

TLR4-activated microglia require IFN-γ to induce severe neuronal dysfunction and death in situ

Microglia (tissue-resident macrophages) represent the main cell type of the innate immune system in the CNS; however, the mechanisms that control the activation of microglia are widely unknown. We systematically explored microglial activation and functional microglia–neuron interactions in organotypic hippocampal slice cultures, i.e., postnatal cortical tissue that lacks adaptive immunity. We applied electrophysiological recordings of local field potential and extracellular K⁺ concentration, immunohistochemistry, design-based stereology, morphometry, Sholl analysis, and biochemical analyses. We show that chronic activation with either bacterial lipopolysaccharide through Toll-like receptor 4 (TLR4) or leukocyte cytokine IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, CD11b and CD68 up-regulation, and proinflammatory cytokine (IL-1β, TNF-α, IL-6) and nitric oxide (NO) release. Notably, these reactive phenotypes only moderately alter intrinsic neuronal excitability and gamma oscillations (30–100 Hz), which emerge from precise synaptic communication of glutamatergic pyramidal cells and fast-spiking, parvalbumin-positive GABAergic interneurons, in local hippocampal networks. Short-term synaptic plasticity and extracellular potassium homeostasis during neural excitation, also reflecting astrocyte function, are unaffected. In contrast, the coactivation of TLR4 and IFN-γ receptors results in neuronal dysfunction and death, caused mainly by enhanced microglial inducible nitric oxide synthase (iNOS) expression and NO release, because iNOS inhibition is neuroprotective. Thus, activation of TLR4 in microglia in situ requires concomitant IFN-γ receptor signaling from peripheral immune cells, such as T helper type 1 and natural killer cells, to unleash neurotoxicity and inflammation-induced neurodegeneration. Our findings provide crucial mechanistic insight into the complex process of microglia activation, with relevance to several neurologic and psychiatric disorders.Microglia are tissue-resident macrophages in the CNS that become activated in most brain disorders, such as bacterial meningoencephalitis, multiple sclerosis, and Alzheimer’s disease (1, 2). Activation of microglia features changes in morphology and receptor expression, antigen presentation, cytokine release, migration, and phagocytosis, and it ranges from proinflammatory and potentially neurotoxic to anti-inflammatory and neuroprotective phenotypes (1, 3, 4). The mechanisms that control the transition of microglia to reactive phenotypes, including the impact on neuronal function, are mostly unknown, however (5–7).Sensing of microbial or modified endogenous ligands by microglia is mediated by innate pattern recognition receptors, such as scavenger receptors and Toll-like receptors (TLRs). A prime example is TLR4, which acts with CD14, MD-2, and lipopolysaccharide (LPS)-binding protein in recognizing LPS, a cell wall component of Gram-negative bacteria (8, 9). TLR4 is also central to microglial recognition of amyloid-β peptide, which is thought to be part of the inflammatory response in Alzheimer’s disease (7, 10).LPS has been widely used to study the molecular mechanisms of microglial activation in inflammatory neurodegeneration (1–3). In primary monocultures and microglia-neuron cultures, LPS exposure alone or in combination with IFN-γ for a “booster” triggers the massive release of proinflammatory and cytotoxic factors, such as TNF-α, IL-6, and nitric oxide (NO), finally resulting in neuronal death (8, 11–18). Similar effects were observed in vivo after intracerebral administration of LPS (19–21). These and other studies have contributed to the concept that microglial TLR4 activation with LPS (i.e., with a single pathogenic stimulus) is sufficient to induce neurodegeneration (22, 23); however, this concept is biologically risky, and has been questioned in some experimental works and reviews (2–4, 11, 24, 25).Most previous studies focused on two aspects of microglial TLR4 activation with LPS: (i) the properties of the reactive microglial phenotype(s) and (ii) the degree of neurodegeneration. For this purpose, either simple culture systems or in vivo models, in which interactions with leukocytes infiltrating from the blood are inevitable, have been used (1, 4). Thus, it is widely unknown how TLR4 and IFN-γ receptor signaling in microglia individually contribute to neurotoxicity and neurodegeneration in situ. This aspect is highly relevant for several neurologic and psychiatric disorders. Moreover, concomitant alterations in neuronal information processing (i.e., dysfunction in excitatory pyramidal cells and inhibitory GABAergic interneurons, including astrocytes) have been little explored (25–27).We rigorously addressed these fundamental questions in postnatal neuronal tissue (1, 4). To mimic microglial confrontation with LPS in situ and, notably, in the absence of infiltrating leukocytes, we used organotypic hippocampal slice cultures that feature highly preserved cytoarchitectures and complex neuronal network functions (5, 28). Microglial interaction with infiltrating T helper type 1 (Th1) cells and/or natural killer (NK) cells was mimicked by recombinant IFN-γ administration.     

TIPE2 protein serves as a negative regulator of phagocytosis and oxidative burst during infection

Phagocytosis and oxidative burst are two major effector arms of innate immunity. Although it is known that both are activated by Toll-like receptors (TLRs) and Rac GTPases, how their strengths are controlled in quiescent and TLR-activated cells is not clear. We report here that TIPE2 (TNFAIP8L2) serves as a negative regulator of innate immunity by linking TLRs to Rac. TLRs control the expression levels of TIPE2, which in turn dictates the strengths of phagocytosis and oxidative burst by binding to and blocking Rac GTPases. Consequently, TIPE2 knockout cells have enhanced phagocytic and bactericidal activities and TIPE2 knockout mice are resistant to bacterial infection. Thus, TIPE2 sets the strengths of phagocytosis and oxidative burst and may be targeted to effectively control infections.Phagocytosis and oxidative burst (or respiratory burst) are two fundamental effector mechanisms of innate immunity that work in concert to eliminate infectious microbes (1, 2). Phagocytosis allows the phagocytes of the immune system (monocytes and granulocytes) to engulf infectious microbes and to contain them in a special vacuole called a phagosome. Oxidative burst in turn injects into the vacuole reactive oxygen species (ROS) (e.g., superoxide radical and hydrogen peroxide) that kill the microbes. Deficiency in either of these innate immune mechanisms leads to immune deficiency and uncontrolled infections (3–6).Both phagocytosis and oxidative burst are controlled by the Rac proteins of the Ras small GTPase superfamily (1–4). There are three mammalian Rac GTPases, which are designated as Rac1, Rac2, and Rac3. Small GTPases are enzymes that hydrolyze GTP. They are active when bound to GTP and inactive when bound to GDP and serve as molecular “on-and-off” switches of signaling pathways that control a wide variety of cellular processes including growth, motility, vesicle trafficking, and death (7). Rac GTPases control phagocytosis by promoting actin polymerization through their effector proteins such as p21-activated kinases (PAKs), WASP family Verprolin homology domain-containing protein (WAVE), and IQ motif containing GTPase-activating protein-1 (IQGAP1) (1). Rac GTPases also mediate ROS production by binding and activating the NADPH oxidase complex through the p67(Phox) protein (1). Rac GTPase deficiency in mice and humans leads to an immune-deficient syndrome, which is characterized by defective phagocytosis and oxidative burst, recurrent infection, and granulomas (3–6).Although quiescent phagocytes are capable of phagocytosis and ROS production, their levels are low. Toll-like receptor (TLR) activation or microbial infection significantly up-regulates these innate immune processes (8–11). However, the mechanisms whereby microbes promote them are not well understood. TIPE2, or tumor necrosis factor-α–induced protein 8 (TNFAIP8)-like 2 (TNFAIP8L2), is a member of the TNFAIP8 family, which is preferentially expressed in hematopoietic cells (12–18). It is significantly down-regulated in patients with infectious or autoimmune disorders (15, 19). The mammalian TNFAIP8 family consists of four members: TNFAIP8, TIPE1, TIPE2, and TIPE3, whose functions are largely unknown (14, 20). We recently generated TIPE2-deficient mice and discovered that TIPE2 plays a crucial role in immune homeostasis (14). We report here that TIPE2 controls innate immunity by targeting the Rac GTPases.     

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3)

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2–glycans with the conserved Lewis^X binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam₂CSK₄ effectively, yet allows SSL3 to bind to an already formed TLR2–Pam₂CSK₄ complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2–lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.In recent years, Staphylococcus aureus has become a major health threat to both humans and domestic animals. It is found as a commensal bacterium in ∼30% of the human population, but when it becomes infectious it can cause a wide diversity of diseases, ranging from mild skin infections to life-threatening invasive conditions such as pneumonia and sepsis (1). Increased antibiotic resistance and a high amount of virulence factors secreted by S. aureus contribute to its emergence as a pathogen. Among these secreted virulence factors are the staphylococcal superantigen-like proteins (SSLs), a family of 14 proteins located on two genomic clusters (2–4). Recently, we and others identified SSL3 as a potent inhibitor of Toll-like receptor 2 (TLR2) (5, 6), an innate immunity receptor that is a dominant factor in immune recognition of S. aureus (7–10).TLR2 belongs to a family of 10 homologous innate immunity receptors that are activated by pathogen-associated molecular patterns (PAMPs) (11). TLR2 binds bacterial lipopeptides and lipoproteins. Subsequent formation of heterodimers with TLR1 or TLR6 leads to MyD88-dependent activation of the NF-κB pathway (12). TLR2 has dual ligand specificity that is determined by its dimerization partner; stimulation by diacyl lipopeptides from Gram-positive bacteria, including S. aureus, induces the formation of heterodimers with TLR6 (13), whereas triacyl lipopeptides from Gram-negative bacteria initiate formation of TLR2–TLR1 dimers (14). The structural basis for lipopeptide specificity was revealed by crystal structures of TLR2–TLR1 and TLR2–TLR6 complexes with their respective lipopeptide analogs Pam₃CSK₄ and Pam₂CSK₄: TLR2 binds two lipid tails in a large hydrophobic pocket, whereas the third lipid tail of triacyl lipopeptides is accommodated by a smaller pocket present in TLR1, but not in TLR6 (15, 16).The family of SSL proteins, including SSL3, share structural similarities to superantigens, but lack superantigenic activity. Interestingly, the functions that have been discovered for SSLs so far have all been linked to immune evasion. SSL5 inhibits neutrophil extravasation (17, 18) and phagocyte function (19, 20), SSL7 binds IgA and inhibits complement (21), and SSL10 inhibits IgG1-mediated phagocytosis (22, 23), blood coagulation (24), and the chemokine receptor CXCR4 (25). In addition to SSL3, also weak TLR2 inhibitory activity was observed for SSL4 (5), but it remains unknown whether that is its dominant function. This variety of immunomodulatory molecules and functions reflects the importance of the different components of our innate immune system in the defense against S. aureus (26).In this study we determined the crystal structures of SSL3 and the SSL3–TLR2 complex. In combination with mutagenesis and binding studies, our data provide a novel working mechanism of a functional TLR2 antagonist.     

Structures and interface mapping of the TIR domain-containing adaptor molecules involved in interferon signaling

Homotypic and heterotypic interactions between Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors (TLRs) and downstream adaptors are essential to evoke innate immune responses. However, such oligomerization properties present intrinsic difficulties in structural studies of TIR domains. Here, using BB-loop mutations that disrupt homotypic interactions, we determined the structures of the monomeric TIR domain-containing adaptor molecule (TICAM)-1 and TICAM-2 TIR domains. Docking of the monomeric structures, together with yeast two hybrid-based mutagenesis assays, reveals that the homotypic interaction between TICAM-2 TIR is indispensable to present a scaffold for recruiting the monomeric moiety of the TICAM-1 TIR dimer. This result proposes a unique idea that oligomerization of upstream TIR domains is crucial for binding of downstream TIR domains. Furthermore, the bivalent nature of each TIR domain dimer can generate a large signaling complex under the activated TLRs, which would recruit downstream signaling molecules efficiently. This model is consistent with previous reports that BB-loop mutants completely abrogate downstream signaling.The extracellular domain of toll-like receptor 4 (TLR4) specifically binds lipopolysaccharides (LPSs) from Gram-negative bacteria, inducing dimerization and leading to the dimerization of cytosolic Toll/interleukin-1 receptor (TIR) domains. This activated conformation of TLR4 recruits the TIR domain of a downstream adaptor molecule, TIR domain-containing adaptor molecule-2 (TICAM-2) [also known as TRIF-related adaptor molecule (TRAM)], that subsequently recruits the TIR domain of another adaptor molecule, TIR domain-containing adaptor molecule-1 (TICAM-1) [also known as TIR domain-containing adaptor inducing IFN-β (TRIF)] (1–3) at endosomes. Eventually this process activates IFN response factors and generates type-I interferons (IFNs) (4–7). Elucidation of the homotypic and heterotypic interactions between TICAM-1 and TICAM-2 is essential for understanding of TLR4-mediated type-I IFN generation (8).A large number of TIR domain structures, including receptors and adaptors, have been determined by X-ray crystallography and NMR. The receptors include TLR1 (9), TLR2 (10), and IL-1R accessory protein-like (IL-1RAPL) (11). Adaptors include myeloid differentiation factor 88 (MyD88) (12) and MyD88 adaptor-like (Mal) (13, 14). In addition, AtTIR (15, 16) derived from Arabidopsis thaliana and PdTIR (17) from bacteria have been solved. Each of these TIR domain structures has a ferredoxin fold with five β-strands (βA–βE), five α-helices (αA–αE), and loops connecting β-strands and α-helices (9). Although homotypic interactions of the TIR domains have been proposed based on the crystal structures, most proposed models have small interacting surfaces, possibly due to crystal contacts. Recently, however, a crystal structure of the TLR10 TIR domain was reported that forms a homotypic dimer mediated by the loop connecting βB and αB (designated “BB-loop”) (18). Interestingly, BB-loop mutations in TLR4 were reported to be dominant-negative and abrogated downstream signaling (19). TICAM-1 and TICAM-2 harboring BB-loop mutations are also dominant-negative and unable to form homotypic interactions (1, 2), reinforcing the importance of BB-loop–mediated homotypic dimer formation in signal propagation.Despite extensive structural studies, it is not known why homotypic interactions are essential for downstream signaling (20–27). To address this issue, it is necessary to discriminate residues required for homotypic and those required for heterotypic interactions. Here, we first determine the structures of the monomeric BB-loop mutants of the TICAM-1 and TICAM-2 TIR domains using NMR. Then, based on the solution structures of the BB-loop mutants, coupled mutagenesis/yeast two-hybrid experiments, and restrained docking calculations, we show that the homotypic interaction of TICAM-2 TIR is essential to form a scaffold for recruiting the TICAM-1 TIR domain.     

Cytokine Sp?tzle binds to the Drosophila immunoreceptor Toll with a neurotrophin-like specificity and couples receptor activation

Drosophila Toll functions in embryonic development and innate immunity and is activated by an endogenous ligand, Spätzle (Spz). The related Toll-like receptors in vertebrates also function in immunity but are activated directly by pathogen-associated molecules such as bacterial endotoxin. Here, we present the crystal structure at 2.35-Å resolution of dimeric Spz bound to a Toll ectodomain encompassing the first 13 leucine-rich repeats. The cystine knot of Spz binds the concave face of the Toll leucine-rich repeat solenoid in an area delineated by N-linked glycans and induces a conformational change. Mutagenesis studies confirm that the interface observed in the crystal structure is relevant for signaling. The asymmetric binding mode of Spz to Toll is similar to that of nerve growth factor (NGF) in complex with the p75 neurotrophin receptor but is distinct from that of microbial ligands bound to the Toll-like receptors. Overall, this study indicates an allosteric signaling mechanism for Toll in which ligand binding to the N terminus induces a conformational change that couples to homodimerization of juxtamembrane structures in the Toll ectodomain C terminus.Both Toll and Spätzle (Spz) were originally identified in screens that detected genes required for dorsoventral axis formation of Drosophila melanogaster embryos (1). Toll is a transmembrane receptor with a composite leucine-rich repeat (LRR) ectodomain, a single-span transmembrane region and an intracellular signaling domain, the Toll/Interleukin-1 receptor domain or TIR. Subsequently, Toll and Spz were shown to mediate antibacterial and antifungal responses in adult flies and larvae (2–5). A related family of molecules, the Toll-like receptors (TLRs), was later discovered in vertebrates, and these receptors also function in innate immunity (6–10). Unlike Drosophila Toll, which is activated by the endogenous ligand Spz, the TLRs are pattern-recognition receptors that respond directly to pathogen-associated molecules such as double-stranded RNA (11, 12). An exception to this mechanism of detection is TLR4, which requires the coreceptor MD-2 to recognize lipolysaccaharides from Gram-negative bacteria (13).Spz resembles mammalian growth factors and particularly the neurotrophin family, such as nerve growth factor (NGF). Spz is synthesized in an inactive form with a signal peptide that drives secretion, an N-terminal prodomain of varying size depending on the splicing process (14) and a C-terminal active fragment of 106 amino acids (C106). The latter adopts a dimeric cystine-knot (CK) structure, a fold very similar to that of NGF (15–19). Spz is activated by maternal signals or immune challenges that trigger specific protease cascades, stimuli that lead to endoproteolytic processing. Before cleavage, the prodomain is thought to mask the receptor-binding site by a mechanism reminiscent of the activation of the chymotrypsin zymogen and coagulogen, a clotting factor from the horseshoe crab (20). Activation-induced proteolysis causes a conformational change that exposes critical determinants for Toll binding on C106 that are masked by the prodomain before proteolysis (20). The prodomain is released from C106 upon Toll binding (21).Previous biochemical studies suggest that initial activation of dToll, like the TLRs, requires ligand-induced dimerization of two receptor ectodomains (21–23). Ion mobility mass spectrometry and electron microscopy reveal that the full-length Toll ectodomain and Spz C106 form both 1:1 and 2:2 complexes although small amounts of a 2:1 stoichiometry can also be detected. This work also indicated that the binding site for Spz is located at the N terminus of the Toll ectodomain. Deletion mutations that truncate the ectodomain are constitutively active (24), and thus the ectodomain has an autoinhibitory function similar to that of vertebrate TLR4 (25). Binding of Spz relieves this inhibition, enabling homodimerization of the receptor and signaling to occur. Several lines of evidence suggest that the extracellular juxtamembrane region is directly involved in receptor homodimerization (24, 26, 27). For example, receptors with mutations of cysteine residues in the juxtamembrane capping structure are constitutively active, and the resultant unpaired cysteine forms intramolecular disulphide bonded dimers. In contrast to the TLRs, dToll signaling displays negative cooperativity so that the receptor responds over a wide range of ligand concentrations (reviewed in ref. 28).In vertebrates, the neurotrophin family have essential developmental functions in neuronal survival, axon targeting, and connectivity and, in adult life, in learning, memory, and cognition (29). The NTs signal through three distinct receptors (NTRs)—p75^NTR, tyrosine receptor kinase (Trk), and Sortilin—and a shared downstream target is the activation of NFκB (30–32). Crystal structures are available for several complexes of vertebrate neurotrophins and their receptors that reveal distinct modes of binding and activation for different combinations of receptors and neurotrophins (33–36). In Drosophila, there are no canonical homologs of these receptors, but a recent study shows that the Toll and Spz paralogues (dToll6/7 and DNT1/2, respectively) fulfill these functions in the insect (37). This finding suggests that Drosophila Toll receptors may be mechanistically more similar to the vertebrate NTRs than to the TLRs. However, other studies show that dToll7 can also mediate antiviral responses to vesicular stomatitis virus and thus has a dual function in the nervous and immune systems (38).To further understand the mechanism of Toll signaling, we crystallized and solved the structure of a complex of the Toll ectodomain and Spz. This structure reveals a mode of binding that is strikingly similar to that of NGF and p75^NTR but different from that observed for the ligands of the mammalian TLRs. This arrangement suggests that ligand binding induces a secondary homodimerization of the receptor ectodomain and provides a molecular explanation for the observed negative allostery of the dToll signaling pathway.     

Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain

Toll/IL-1R resistance (TIR) domain–containing adapter-inducing IFN-β (TRIF) is a Toll-like receptor (TLR) adapter that mediates MyD88-independent induction of type I interferons through activation of IFN regulatory factor 3 and NFκB. We have examined peptides derived from the TRIF TIR domain for ability to inhibit TLR4. In addition to a previously identified BB loop peptide (TF4), a peptide derived from putative helix B of TRIF TIR (TF5) strongly inhibits LPS-induced cytokine and MAPK activation in wild-type cells. TF5 failed to inhibit LPS-induced cytokine and kinase activation in TRIF-deficient immortalized bone-marrow–derived macrophage, but was fully inhibitory in MyD88 knockout cells. TF5 does not block macrophage activation induced by TLR2, TLR3, TLR9, or retinoic acid-inducible gene 1/melanoma differentiation-associated protein 5 agonists. Immunoprecipitation assays demonstrated that TF4 binds to TLR4 but not TRIF-related adaptor molecule (TRAM), whereas TF5 binds to TRAM strongly and TLR4 to a lesser extent. Although TF5 prevented coimmunoprecipitation of TRIF with both TRAM and TLR4, site-directed mutagenesis of the TRIF B helix residues affected TRIF–TRAM coimmunoprecipitation selectively, as these mutations did not block TRIF–TLR4 association. These results suggest that the folded TRIF TIR domain associates with TRAM through the TRIF B helix region, but uses a different region for TRIF–TLR4 association. The B helix peptide TF5, however, can associate with either TRAM or TLR4. In a mouse model of TLR4-driven inflammation, TF5 decreased plasma cytokine levels and protected mice from a lethal LPS challenge. Our data identify TRIF sites that are important for interaction with TLR4 and TRAM, and demonstrate that TF5 is a potent TLR4 inhibitor with significant potential as a candidate therapeutic for human sepsis.Toll-like receptors (TLRs) initiate innate immune responses by recognizing specific pathogen-associated molecules; for example, TLR4 recognizes lipopolysaccharides (LPSs) of Gram-negative bacteria (1, 2). Ligand recognition induces dimerization of cytoplasmic Toll/IL-1R resistance (TIR) domains of two receptor molecules and causes recruitment of intracellular TIR domain-containing adapters. Four adapter proteins participate in TLR4 signaling: myeloid differentiation factor 88 (MyD88) (3), TIR domain-containing adapter protein, also known as MyD88-adapter-like (TIRAP–Mal) (4, 5), TIR domain–containing adapter-inducing IFN-β, also known as TLR adaptor molecule 1 (TRIF–TICAM-1) (6, 7), and TRIF-related adaptor molecule also known as TLR adaptor molecule 2 (TRAM–TICAM-2) (8, 9). TIRAP–Mal is important for MyD88 recruitment to the signaling complex located at the plasma membrane to initiate early NF-κB and mitogen-activated protein kinase (MAPK) activation and induce “MyD88-dependent” proinflammatory cytokines, such as TNF-α and IL-1β (4, 5, 10). TRAM is important for TRIF recruitment to the endosomally located TLR4 signaling complexes to activate IFN regulatory factor 3 (IRF3) and induce IRF3-dependent cytokines, such as IFN-β and RANTES (regulated upon activation normal T-cell expressed and secreted) (8, 9, 11).A typical TIR domain consists of the central five stranded parallel β sheets (the strands are designated as βA–βE) surrounded by 5 α-helices (i.e., αA–αE) (12, 13). The TIRAP–Mal TIR domain has an atypical fold compared with other resolved mammalian TIR structures in that the position of its β-strand B is shifted by 12–18 amino acids toward the C terminus, so that TIRAP TIR does not have a helix B but has an unusually long AB loop (14, 15). Structures of the TIR domains of TLR4, TRIF, and TRAM have not been yet resolved. The TIR domain is a key structural feature present in all TLRs and TLR adapter proteins. TIR domains mediate transient homotypic or heterotypic protein interactions required for agonist-driven assembly of TLR signaling complexes (13, 16, 17). Multiple interactions of TIR domains of TLRs and TLR adapters are required to mediate adapter recruitment and stabilize initial complex (18–20). It has been proposed that TLR4 activation leads to formation of several compositionally distinct complexes. Kagan et al. proposed that TLR4 engages TIRAP–MyD88 and TRAM–TRIF sequentially at distinct cellular locations (11), thus implying that the two sets of adapters may compete for the same binding site at the TLR4 homodimer. However, it remains unclear how exactly the four adapters interact with each other and TLR4 to orchestrate TLR4 signaling.The presumed mechanism of signaling inhibition by a decoy peptide is that the peptide competes with its prototype protein for the prototype’s docking site and thereby prevents a protein–protein interaction required for signaling (19). In this study, we have examined cell-permeable decoy peptides derived from the TIR domain of TRIF. Two peptides, TF4 and TF5, from the second loop (BB loop) and the second helical region (helix B) of the TRIF TIR, respectively, potently inhibited LPS-induced activation of MAPKs and induction of MyD88-dependent and TRIF-dependent cytokines in wild-type macrophages. TF5 did not inhibit TLR4 signaling in TRIF^−/− immortalized bone-marrow–derived macrophages (iBMDMs) but did exhibit full activity in the MyD88^−/− cells. TF5 inhibits TLR4-driven macrophage signaling at a lower dose in vitro compared with TF4 and binds to both TRAM and TLR4, whereas TF4 targets TLR4 but not the TRAM TIR. In a mouse model of TLR4-driven inflammation, TF5 potently decreased the systemic cytokine levels induced in mice by a sublethal LPS dose, and dramatically improved survival of mice challenged with a lethal LPS dose.