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.     

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.     

Multifaceted contribution of the TLR4-activated IRF5 transcription factor in systemic sclerosis

Systemic sclerosis (SSc) is a multisystem autoimmune disorder with clinical manifestations resulting from tissue fibrosis and extensive vasculopathy. A potential disease susceptibility gene for SSc is IFN regulatory factor 5 (IRF5), whose SNP is associated with milder clinical manifestations; however, the underlying mechanisms of this association remain elusive. In this study we examined IRF5-deficient (Irf5^−/−) mice in the bleomycin-treated SSc murine model. We show that dermal and pulmonary fibrosis induced by bleomycin is attenuated in Irf5^−/− mice. Interestingly, we find that multiple SSc-associated events, such as fibroblast activation, inflammatory cell infiltration, endothelial-to-mesenchymal transition, vascular destabilization, Th2/Th17 skewed immune polarization, and B-cell activation, are suppressed in these mice. We further provide evidence that IRF5, activated by Toll-like receptor 4 (TLR4), binds to the promoters of various key genes involved in SSc disease pathology. These observations are congruent with the high level of expression of IRF5, TLR4, and potential endogenous TLR4 ligands in SSc skin lesions. Our study sheds light on the TLR4-IRF5 pathway in the pathology of SSc with clinical implications of targeting the IRF5 pathways in the suppression of disease development.Systemic sclerosis (SSc) is a multisystem connective tissue disease characterized by immune abnormalities, vasculopathy, and extensive tissue fibrosis (1). Based on the results of etiological and genetic studies, the conventional wisdom is that SSc is caused by a complex interplay between genetic factors and environmental influences. For instance, the biggest risk factor for SSc is family history (2). On the other hand, concordance for SSc is around 5% in twins and is similar in monozygotic and dizygotic twins, whereas antinuclear antibodies are detected more frequently in the healthy monozygotic twin sibling than in the healthy dizygotic twin sibling of an SSc patient (3). In addition, most SSc susceptibility genes are HLA haplotypes and non-HLA immune-related genes that are shared by other collagen diseases (4). Therefore, genetic factors are likely associated with autoimmunity, increasing the susceptibility to autoimmune diseases including SSc, but additional environmental factors are required to induce clinically definite SSc in genetically predisposed individuals. Despite these etiological and genetic data, the entire process of the SSc development and pathogenesis remains elusive.Therefore it is important to elucidate the molecular mechanism(s) underlying SSc pathogenesis. In this regard, much attention has been focused recently on the innate immune signaling via Toll-like receptors (TLRs) in various pathological conditions. For instance, fibroblasts and endothelial cells in SSc lesional skin highly express TLR4, originally identified as the receptor for bacterial LPS, and TLR4 signaling amplifies the sensitivity to TGF-β in dermal fibroblasts (5–7). It also was shown that dermal and lung fibrosis is attenuated in bleomycin (BLM)-treated TLR4-deficient mice (7). Endogenous potential TLR4 ligands are up-regulated in SSc lesional skin (5–7), and serum levels correlate with severe organ involvement and immunological abnormalities (8, 9). Therefore, the TLR4 signaling pathway is suspected to play a central role in the SSc pathogenesis.Although how the TLR4 signaling pathway contributes to SSc pathogenesis remains enigmatic, it is interesting that several independent case-control and genome-wide association studies identify IFN regulatory factor 5 (IRF5), a member of the IFN regulatory factor (IRF) family, as an SSc susceptibility gene (10–15). IRFs were identified primarily in the research of the type I IFN system and have been shown to have functionally diverse roles in the regulation of the innate and adaptive immune responses (16). Reflecting such property of IRFs, SNPs of IRFs have been linked to the development of various immune and inflammatory disorders. IRF5 is of particular interest, being implicated in multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and SSc (14). Thus far an association of certain SNPs within the IRF5 promoter with the risk and severity of SSc has been reported (10–15), but whether and how IRF5 is activated to contribute to disease development remains unknown.Stimulation of TLRs triggers the activation of myeloid differentiation factor 88 (MyD88)-dependent and/or independent pathways (16). IRF5 is activated via the MyD88 pathway in dendritic cells and macrophages (17). TLR-activated IRF5 mediates the induction of genes IL-6, IL-12, and TNF-α (17). Hence, an intriguing possibility is that TLR4-mediated activation of IRF5 is involved in SSc. We therefore studied the role of IRF5 in the regulation of genes associated with the susceptibility to and the severity of SSc using IRF5-deficient mice in the context of TLR4 signaling. We show that IRF5, activated by TLR4, binds to the promoters of various key genes involved in the disease symptoms. We discuss our findings in terms of the complexity of SSc and its clinical implications.     

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.     

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.     

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.     

RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition

The pronecrotic kinase, receptor interacting protein (RIP1, also called RIPK1) mediates programmed necrosis and, together with its partner, RIP3 (RIPK3), drives midgestational death of caspase 8 (Casp8)-deficient embryos. RIP1 controls a second vital step in mammalian development immediately after birth, the mechanism of which remains unresolved. Rip1^−/− mice display perinatal lethality, accompanied by gross immune system abnormalities. Here we show that RIP1 K45A (kinase dead) knockin mice develop normally into adulthood, indicating that development does not require RIP1 kinase activity. In the face of complete RIP1 deficiency, cells develop sensitivity to RIP3-mixed lineage kinase domain-like–mediated necroptosis as well as to Casp8-mediated apoptosis activated by diverse innate immune stimuli (e.g., TNF, IFN, double-stranded RNA). When either RIP3 or Casp8 is disrupted in combination with RIP1, the resulting double knockout mice exhibit slightly prolonged survival over RIP1-deficient animals. Surprisingly, triple knockout mice with combined RIP1, RIP3, and Casp8 deficiency develop into viable and fertile adults, with the capacity to produce normal levels of myeloid and lymphoid lineage cells. Despite the combined deficiency, these mice sustain a functional immune system that responds robustly to viral challenge. A single allele of Rip3 is tolerated in Rip1^−/−Casp8^−/−Rip3^+/− mice, contrasting the need to eliminate both alleles of either Rip1 or Rip3 to rescue midgestational death of Casp8-deficient mice. These observations reveal a vital kinase-independent role for RIP1 in preventing pronecrotic as well as proapoptotic signaling events associated with life-threatening innate immune activation at the time of mammalian parturition.Receptor interacting protein (RIP) kinase RIP1 (RIPK1) functions as an essential adapter in a number of innate immune signal transduction pathways, including those initiated by Toll-like receptor (TLR)3, TLR4, and retinoic acid-inducible gene 1 (RIG-I)-like receptors, in addition to death receptors (1–4). Signaling via these pathways bifurcates at the level of RIP1 to produce opposing outcomes, a prosurvival inflammatory response counterbalanced by extrinsic cell death signaling that drives either apoptosis or necroptosis. Despite the normal development of many organs and neuromuscular architecture, RIP1-null mice die within a few days of birth with signs of edema as well as significant levels of cell death within lymphoid tissues, particularly immature thymocytes (5). Although TNF-signaling contributes to this perinatal death (6) and implicates the prosurvival role of RIP1 in activating nuclear factor κB (NF-κB) (5), the precise mechanism responsible for developmental failure of RIP1-deficient mice remains unresolved. It seems likely that dysregulation of additional signaling pathways contributes to this phenotype, given that deficiency in TNF receptor 1 (TNFR1) only modestly extends the lifespan of RIP1-null mice and deficiency in TNFR2 only rescues thymocytes from death (7).RIP1 orchestrates assembly of distinct signaling platforms via two C-terminal protein–protein binding domains: a death domain and a RIP homotypic interaction motif (RHIM) (3, 4). This unique architecture facilitates convergent death domain-dependent and RHIM-dependent pathways. RIP1 partners with death domain-containing proteins, particularly fas-associated death domain protein (FADD), as well as RHIM-containing proteins, such as the pronecrotic kinase RIP3 and the TLR3/TLR4 adapter TIR-domain–containing adapter-inducing IFN (TRIF) (8, 9). RIP1 is essential for TNF-induced necroptosis but dispensable for other forms of RIP3 kinase-dependent death (10, 11). Oligomerization of RIP1 through either domain promotes activation of its N-terminal serine/threonine kinase and triggers either of two distinct cell death pathways: (i) apoptosis following assembly of a cytosolic FADD–Casp8–cellular FLICE-like inhibitory protein (cFLIP)-containing complex or (ii) necroptosis via RIP3-dependent, mixed lineage kinase domain-like (MLKL)-mediated membrane permeabilization (1–4).In addition to death, RIP1 activation downstream of either TNFR1 or TNFR2 facilitates prosurvival NF-κB gene expression contingent on the balance of ubiquitination and deubiquitination (12). In this context, deubiquitination converts RIP1 into a death-inducing adapter within the TNFR-signaling complex (12). RIP1 remains a component of a death receptor-free cytosolic complex, termed complex II (also called the ripoptosome) (1–3), together with FADD, Casp8, and cFLIP where cFLIP levels control Casp8 activation (13) and death (14). When Casp8 or FADD are absent or Casp8 activity is inhibited (14–17), RIP1 mediates RHIM-dependent recruitment of RIP3. Then, RIP1 kinase activity facilitates RIP3 kinase-dependent phosphorylation of MLKL to drive necroptosis (18, 19). Importantly, basal Casp8 activity conferred by cFLIP blocks this process (14), and in vivo, this translates into a unique requirement for Casp8 to prevent RIP3-dependent embryonic lethality and tissue inflammation triggered by Casp8 or FADD compromise (14–17). Recently, the importance of Casp8 suppression of necroptosis has been extended to diverse innate signaling pathways, including those activated by TLR3 as well as type I or II interferon (IFN) (11, 20, 21), broadening a concept that first emerged in death receptor signaling (3, 4). Once TLR3 becomes activated, the adapter protein TRIF recruits RIP1 or RIP3 via RHIM interactions (8). In this context, the RIP1 death domain ensures the suppression of necrotic death by recruiting FADD, Casp8, and cFLIP. Necroptosis is unleashed whenever Casp8 or FADD is compromised. Likewise, IFN activation of protein kinase R sets up a similar relationship with the FADD–Casp8–cFLIP–RIP1 complex (21). Thus, innate immunity elicits dueling signals that both potentiate and suppress programmed necrosis.In this study, we implicate multiple innate immune signaling pathways in the death of RIP1-deficient mice. Once dysregulated by disruption of RIP1, RIP3-mediated necroptosis and Casp8-dependent apoptosis contribute to death at the time of birth. Our observations bring to light the consequences of diverse innate immune stimuli arising from TNF, IFN, and/or nucleic acids that play out during mammalian parturition. RIP1 plays a vital role suppressing cell death consequences of this innate signaling. RIP3 and Casp8 must be eliminated to rescue RIP1-null mice from perinatal death and produce fully viable, fertile, and immunocompetent triple-knockout (TKO) mice.