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.     

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.     

Macrophages eat cancer cells using their own calreticulin as a guide: Roles of TLR and Btk

Macrophage-mediated programmed cell removal (PrCR) is an important mechanism of eliminating diseased and damaged cells before programmed cell death. The induction of PrCR by eat-me signals on tumor cells is countered by don’t-eat-me signals such as CD47, which binds macrophage signal-regulatory protein α to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Here we demonstrate that the activation of Toll-like receptor (TLR) signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance PrCR. Bruton’s tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be an eat-me signal on cancer cells, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.Programmed cell removal (PrCR) is a process of macrophage-mediated immunosurveillance by which target cells are recognized and phagocytosed (1). PrCR previously was known to be a key step concurrent with programmed cell death for the clearance of apoptotic cells, but when apoptosis is blocked, PrCR of neutrophils that are living (because of the enforced expression of bcl2) occurs precisely at the same time that PrCR removes dying wild-type neutrophils (2). Recently a role for PrCR in eliminating living tumor cells has been revealed (1). Several studies have indicated a crucial function of CD47 as an antiphagocytic don''t-eat-me signal dominating over PrCR (3–10). During cancer development, tumor cells up-regulate CD47, which protects them from PrCR (1, 3, 4, 6). Blockade of the interaction between CD47 on target cells and its receptor, signal-regulatory protein α (SIRPα), on macrophages elicits efficient PrCR of cancer cells but not of most normal cells in vitro and in vivo (Fig. 1A) (1, 3). When CD47 is blocked, cancer cells, but not normal cells, are phagocytosed because prophagocytic eat-me signals such as calreticulin (CRT) are commonly expressed on many leukemias, lymphomas, and solid tumors (Fig. 1A) (11). CRT normally is an endoplasmic reticulum (ER) protein possessing ER retention KDEL sequences but can be released to the cell surface in many instances of cell damage by cytotoxic drugs or inflammation and is recognized by macrophage LRP1/CD91 during phagocytosis of apoptotic cells (12, 13). Bruton’s tyrosine kinase (Btk) is a member of the Tec nonreceptor protein tyrosine kinase family, which plays a crucial role in the regulation of the innate immune response (14, 15). A defect of Btk leads to immunodeficiencies including X-linked hypo- or agammaglobulinemia (16–18), presumably caused by the blockade of B-cell development and perhaps related to inefficient clearance of defective B-lineage cells as well (19). Thus far, however, little is known about the molecular mechanisms by which macrophages recognize and phagocytose living cancer cells. We show here that macrophages express CRT and that Toll-like receptor (TLR) signaling through Btk results in its trafficking to the cell surface, where it can be used to mediate PrCR of appropriate tumor cells.Open in a separate windowFig. 1.Activation of TLR signaling leads to enhanced PrCR of living cancer cells. (A, Left) Schematic showing PrCR of living tumor cells by macrophages. Blockade of CD47 leads to an imbalance of eat-me over don’t-eat-me pathways, which elicits phagocytosis of tumor cells, either Fc-dependent (elicited by Fc–FcR interaction) or Fc-independent (labeled in red, representing cancer-specific eat-me signals other than Fc). (Right) A phagocytosis assay showing blockade of CD47-induced phagocytosis, with SW620 cells [control IgG-treated, anti-CD47 antibody (B6H12)–treated, or CD47^KO] as target cells and BMDMs from RAG2^−/−, γc^−/− mice. Fc receptor blocker (FcRB) reversed phagocytosis of B6H12-treated cells to the same level as inf CD47^KO cells. **P < 0.01, t test; ns, not significant. (B) A phagocytosis assay showing a screen of TLR agonists, with SW620 cells [PBS-treated, anti-CD47 antibody (Hu5F9-G4)–treated, or CD47^KO] as target cells and BMDMs from BALB/c mice. TLR agonists used in the screen were Pam3CSK4 (Pam, TLR1/2), heat-killed Listeria monocytogenes (HKLM, TLR2), poly (I:C) HMW [poly (I:C), TLR3)], lipopolysaccharide (LPS, TLR4), flagellin from Salmonella typhimurium (FLA-ST, TLR5), Pam2CGDPKHPKSF (FSL-1, TLR6/2), imiquimod (Imi, TLR7), and class B CpG oligonucleotide (ODN 1826, TLR9). Dashed lines indicate twofold phagocytosis of each condition [PBS-treated, anti-CD47 antibody (Hu5F9-G4)-treated, or CD47^KO] in the control macrophage group. Error bars represent SD.     

Osteopontin expression by CD103? dendritic cells drives intestinal inflammation

Intestinal CD103⁻ dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103⁻ DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103⁻ DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103⁻ DCs resulted in attenuated colitis in comparison to transfer of WT CD103⁻ DCs, whereas transgenic CD103⁻ DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103⁻ DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103⁻ DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103⁻ DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103⁻ DCs and their function during inflammatory bowel disease.Inflammatory bowel diseases (IBDs), including Crohn disease (CD) and ulcerative colitis (UC), are caused by excessive inflammatory responses to commensal microflora and other antigens present in the intestinal lumen (1). Intestinal dendritic cells (DCs) contribute to these inflammatory responses during human IBD, as well as in murine colitis models (2). DCs that reside in draining mesenteric lymph nodes (MLNs) are also crucial mediators of colitis induction (3) and may be grouped based on their surface CD103 (integrin αE) expression as CD11c^highCD103⁺ (CD103⁺ DCs) and CD11c^highCD103⁻ (CD103⁻ DCs) (4–6). CD103⁺ DCs are considered important mediators of gut homeostasis in steady state (4, 5, 7–9), and their tolerogenic properties are conserved between mice and humans (5). However, their role during intestinal inflammation is not well defined. Instead, CD103⁻ DC function has been described mostly during chronic experimental colitis (10–12). These cells secrete IL-23, IL-6, and IL-12 (10–12), contributing to the development of T helper (Th) 17 and Th1 cells, and are highly inflammatory during CD4⁺ T-cell transfer colitis (12) and during 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis (11). MLN CD103⁻ DCs cultured in the presence of LPS, a Toll-like receptor (TLR) 4 agonist, or R848, a TLR7 agonist, express higher levels of TNF-α and IL-6 (7, 12). In fact, these cells secrete IL-23 and IL-12 even in the absence of TLR stimulation (10). Both MLN CD103⁻ and CD103⁺ DC subsets are present in acute colitis (11, 13); however, their function, as well as their cytokine profile, during this phase of disease, reflecting colitis initiation, remains unknown.Recent studies suggest a proinflammatory role for the cytokine osteopontin (Opn) in TNBS- and dextran sulfate sodium (DSS)-induced colitis (14, 15), which are the models for CD and UC, respectively. Opn is expressed by DCs and other immune cell types, such as lymphocytes, during autoimmune responses (16–22), and its expression by DCs during autoimmunity contributes to disease severity (17–19, 21, 23). In addition, Opn expression is highly up-regulated in intestinal immune and nonimmune cells and in the plasma of patients with CD and UC (24–29), as well as in the colon and plasma of mice with experimental colitis (14, 15, 27, 30). Increased plasma Opn levels are related to the severity of CD inflammation (29), and certain Opn gene (Spp1) haplotypes are modifiers of CD susceptibility (31), indicating that Opn could be used as an IBD biomarker (27). In general, Opn affects DC biology during several inflammatory conditions (17–21, 32–37) and could be a potential therapeutic target in IBD.In this study, we initially asked whether Opn was expressed by MLN CD103⁻ and CD103⁺ DCs during colitis. We found that CD103⁻ DCs express excessive levels of Opn in addition to other proinflammatory cytokines. Conversely, CD103⁺ DCs express profoundly lower levels of Opn and are noninflammatory. Using adoptive transfer of purified specific DC subsets, we determined that MLN CD103⁻ DCs are critical mediators of acute intestinal inflammation and that their Opn expression is essential for their proinflammatory properties in both acute and chronic colitis. Furthermore, Opn-deficient and Opn-neutralized mice developed significantly milder disease. In addition, we constructed transgenic (Tg) mice overexpressing Opn only in DCs. These mice developed exaggerated colitis, and adoptive transfer of their CD103⁻ DCs into recipient mice dramatically exacerbated disease. Because Opn protein contains several domains interacting with various receptors, we defined a specific Opn domain significant for inducing proinflammatory properties in CD103⁻ DCs. Blockade of the interaction of this Opn domain [containing functional Ser-Leu-Ala-Tyr-Gly-Leu-Arg (SLAYGLR) sequence] with integrin α9 expressed on CD103⁻ DCs abrogated their proinflammatory profile and colitogenic effects in vivo.     

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.     

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.     

Hyperglycemia in rodent models of type 2 diabetes requires insulin-resistant alpha cells

To determine the role of glucagon action in diet-induced and genetic type 2 diabetes (T2D), we studied high-fat-diet–induced obese (DIO) and leptin receptor-defective (LepR^−/−) rodents with and without glucagon receptors (GcgRs). DIO and LepR^−/−,GcgR^+/+ mice both developed hyperinsulinemia, increased liver sterol response element binding protein 1c, and obesity. DIO GcgR^+/+ mice developed mild T2D, whereas LepR^−/−,GcgR^+/+ mice developed severe T2D. High-fat–fed (HFF) glucagon receptor-null mice did not develop hyperinsulinemia, increased liver sterol response element binding protein 1c mRNA, or obesity. Insulin treatment of HFF GcgR^−/− to simulate HFF-induced hyperinsulinemia caused obesity and mild T2D. LepR^−/−,GcgR^−/− did not develop hyperinsulinemia or hyperglycemia. Adenoviral delivery of GcgR to GcgR^−/−,LepR^−/− mice caused the severe hyperinsulinemia and hyperglycemia of LepR^−/− mice to appear. Spontaneous disappearance of the GcgR transgene abolished the hyperinsulinemia and hyperglycemia. In conclusion, T2D hyperglycemia requires unsuppressible hyperglucagonemia from insulin-resistant α cells and is prevented by glucagon suppression or blockade.The prevalence of type 2 diabetes (T2D) in the United States was 29.1 million in 2012, and 37% of adults were identified as prediabetic (1). T2D is now present on every continent (2). Despite the magnitude of this threat to world physical and fiscal health, our understanding of the pathogenic pathway is vague and is based largely on epidemiologic correlations. For example, the correlation between T2D and obesity is so high that most obese Americans can be considered prediabetic, but the precise mechanism of this relationship is unknown. Although the “lipotoxic” effects of ectopic lipids were first suggested in 1994 (3) to link diet-induced obesity to T2D and other components of the metabolic syndrome (3–11), the relationship between IR and T2D is still poorly understood. Proposed hypothetical links range from beta cell “glucotoxicity” (12) to the action of modifier genes (13) to failure of redox control (14).It has recently been shown that glucagon receptor-null mice remain normoglycemic and nonketotic despite total insulin deficiency but that transduction of a glucagon receptor cDNA into their liver makes them severely diabetic (15, 16). This proves that, whether or not insulin action is present, suppression of glucagon action prevents hyperglycemia. It has long been known that insulin suppression of glucagon regulates alpha cell secretion (17, 18). Although the presence of hyperglucagonemia was established unequivocally in type 1 diabetes (T1D) (15, 16), direct evidence that it is essential for the hyperglycemia of T2D is lacking. However, it has long been known that glucagon is elevated in T2D (17, 19, 20) and is resistant to suppression by insulin.     

B and T lymphocyte attenuator inhibits LPS-induced endotoxic shock by suppressing Toll-like receptor 4 signaling in innate immune cells

Although innate immune responses are necessary for the initiation of acquired immune responses and the subsequent successful elimination of pathogens, excessive responses occasionally result in lethal endotoxic shock accompanied by a cytokine storm. B and T lymphocyte attenuator (BTLA), a coinhibitory receptor with similarities to cytotoxic T-lymphocyte antigen (CTLA)-4 and programmed death (PD)-1, is expressed in not only B and T cells but also dendritic cells (DCs) and macrophages (Mϕs). Recently, several studies have reported that BTLA-deficient (BTLA^−/−) mice show enhanced pathogen clearance compared with WT mice in early phase of infections. However, the roles of BTLA expressed on innate cells in overwhelming and uncontrolled immune responses remain unclear. Here, we found that BTLA^−/− mice were highly susceptible to LPS-induced endotoxic shock. LPS-induced TNF-α and IL-12 production in DCs and Mϕs was significantly enhanced in BTLA^−/− mice. BTLA^−/− DCs also produced high levels of TNF-α on stimulation with Pam3CSK4 but not poly(I:C) or CpG, suggesting that BTLA functions as an inhibitory molecule on Toll-like receptor signaling at cell surface but not endosome. Moreover, BTLA^−/− DCs showed enhanced MyD88- and toll/IL-1R domain-containing adaptor inducing IFN (TRIF)-dependent signaling on LPS stimulation, which is associated with impaired accumulation of Src homology 2-containing protein tyrosine phosphatase in lipid rafts. Finally, we found that an agonistic anti-BTLA antibody rescued mice from LPS-induced endotoxic shock, even if the antibody was given to mice that had developed a sign of endotoxic shock. These results suggest that BTLA directly inhibits LPS responses in DCs and Mϕs and that agonistic agents for BTLA might have therapeutic potential for LPS-induced endotoxic shock.Septic shock is a life-threatening disease, which is caused by bacterial infection, especially with Gram-negative bacteria (1, 2). Toll-like receptor 4 (TLR4), one of representative pattern recognition receptors, recognizes LPS from Gram-negative bacteria and transduces signals in innate cells, such as macrophages (Mϕs) and dendritic cells (DCs), for the production of proinflammatory cytokines and chemokines (2–4). These innate responses are necessary for the initiation of acquired immune responses and subsequent successful elimination of bacteria. However, excessive innate immune responses occasionally result in a cytokine storm that is a potentially fatal immune reaction consisting of a positive feedback loop between highly elevated levels of various cytokines and immune cells, which leads to lethal endotoxic shock within a few days (1, 3, 5–8). However, lethal endotoxic shock is difficult to control by inhibitors for a particular cytokine (2, 7), and thus, novel therapeutic strategies for lethal endotoxic shock are desired.B and T lymphocyte attenuator (BTLA; CD272) is the third inhibitory coreceptor, which has been identified as an inhibitory coreceptor expressed on CD4⁺ T cells and B cells with similarities to CTLA-4 and PD-1 (9). Thereafter, accumulating evidence has revealed that BTLA is expressed on not only CD4⁺ T and B cells but also a wide range of hematopoietic cells, including CD8⁺ T cells, natural killer T cells, natural killer cells, Mϕs, and DCs at various levels (10). The ligand for BTLA is the TNF receptor family member Herpesvirus entry mediator (HVEM), which is broadly expressed on hematopoietic cells, including T cells, Mϕs, and DCs (10). Ligation of BTLA by HVEM induces the recruitment of SHP-1/SHP-2 and then attenuates cell activation (9–11). Analyses of BTLA-deficient (BTLA^−/−) mice have revealed that BTLA plays inhibitory roles in a variety of disease models, including experimental autoimmune encephalomyelitis (9), partially MHC-mismatched cardiac allograft (12), experimental colitis (13), and experimental hepatitis (14). We have also shown that the deficiency of BTLA spontaneously causes the breakdown of self-tolerance, resulting in the development of an autoimmune hepatitis-like disease and lymphocytic infiltration in multiple organs in aged mice (15). However, the administration of an agonistic anti-BLTA antibody has been shown to prevent graft-versus-host disease (16) and hapten-induced contact hypersensitivity (17). These results suggest that BTLA plays an important role in the homeostasis of acquired immune responses.In addition to the role of BTLA in acquired immune responses, recent studies have shown that BTLA also plays a role in immune responses against infectious pathogens. Sun et al. (18) have shown that BTLA^−/− mice exhibit significantly higher bacterial clearance compared with WT mice in the early phase of bacterial infection. Shubin et al. (19) have also shown that BTLA^−/− mice exhibited a higher rate of survival and protection from cecal ligation and puncture. Moreover, Adler et al. (20) have shown that BTLA^−/− mice exhibit strongly enhanced parasite clearance and that the increased clearance is seen before the onset of acquired immune responses. These findings suggest that BTLA is involved in the clearance of pathogens in the early phase of immune responses and that BTLA expressed on innate cells might be involved in the process. However, the role of BTLA in overwhelming and uncontrolled immune responses that lead to endotoixic shock remains unclear.In this study, we examined the role of BTLA in innate immune responses and found that BTLA signaling inhibited LPS-induced endotoxic shock and proinflammatory cytokine production from innate cells. We also found that BTLA signaling inhibited both LPS-induced MyD88- and TRIF-dependent pathways in DCs, possibly by inducing the recruitment of SHP-2 into lipid rafts. We also showed that an agonistic anti-BTLA antibody had therapeutic potential for endotoxic shock. Our results highlight the importance of BTLA in innate immune responses.     

Effects of lymphocyte profile on development of EBV-induced lymphoma subtypes in humanized mice

Epstein-Barr virus (EBV) infection causes both Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). The present study reveals that EBV-induced HL and NHL are intriguingly associated with a repopulated immune cell profile in humanized mice. Newborn immunodeficient NSG mice were engrafted with human cord blood CD34⁺ hematopoietic stem cells (HSCs) for a 8- or 15-wk reconstitution period (denoted ^8whN and ^15whN, respectively), resulting in human B-cell and T-cell predominance in peripheral blood cells, respectively. Further, novel humanized mice were established via engraftment of hCD34⁺ HSCs together with nonautologous fetal liver-derived mesenchymal stem cells (MSCs) or MSCs expressing an active notch ligand DLK1, resulting in mice skewed with human B or T cells, respectively. After EBV infection, whereas NHL developed more frequently in B-cell–predominant humanized mice, HL was seen in T-cell–predominant mice (P = 0.0013). Whereas human splenocytes from NHL-bearing mice were positive for EBV-associated NHL markers (hBCL2⁺, hCD20⁺, hKi67⁺, hCD20⁺/EBNA1⁺, and EBER⁺) but negative for HL markers (LMP1⁻, EBNA2⁻, and hCD30⁻), most HL-like tumors were characterized by the presence of malignant Hodgkin’s Reed–Sternberg (HRS)-like cells, lacunar RS (hCD30⁺, hCD15⁺, IgJ⁻, EBER⁺/hCD30⁺, EBNA1⁺/hCD30⁺, LMP⁺/EBNA2⁻, hCD68⁺, hBCL2⁻, hCD20^-/weak, Phospho STAT6⁺), and mummified RS cells. This study reveals that immune cell composition plays an important role in the development of EBV-induced B-cell lymphoma.Epstein Barr virus (EBV) infects human B lymphocytes and epithelial cells in >90% of the human population (1, 2). EBV infection is widely associated with the development of diverse human disorders that include Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphomas (NHL), including diffused large B-cell lymphoma (DLBCL), follicular B-cell lymphoma (FBCL), endemic Burkitt’s lymphoma (BL), and hemophagocytic lymphohistiocytosis (HLH) (3).HL is a malignant lymphoid neoplasm most prevalent in adolescents and young adults (4–6). Hodgkin/Reed–Sternberg (HRS) cells are the sole malignant cells of HL. HRS cells are characterized by CD30⁺/CD15⁺/BCL6⁻/CD20^+/− markers and appear large and multinucleated owing to multiple nuclear divisions without cytokinesis. Although HRS cells are malignant in the body, surrounding inflammatory cells greatly outnumber them. These reactive nonmalignant inflammatory cells, including lymphocytes, histiocytes, eosinophils, fibroblasts, neutrophils, and plasma cells, compose the vast majority of the tumor mass. The presence of HRS cells in the context of this inflammatory cellular background is a critical hallmark of the HL diagnosis (4). Approximately 50% of HL cases are EBV-associated (EBVaHL) (7–11). EBV-positive HRS cells express EBV latent membrane protein (LMP) 1 (LMP1), LMP2A, LMP2B, and EBV nuclear antigen (EBNA) 1 (EBNA1), but lack EBNA2 (latency II marker) (12). LMP1 is consistently expressed in all EBV-associated cases of classical HL (13, 14). LMP1 mimics activated CD40 receptors, induces NF-κB, and allows cells to become malignant while escaping apoptosis (15).The etiologic role of EBV in numerous disorders has been studied in humanized mouse models in diverse experimental conditions. Humanized mouse models recapitulate key characteristics of EBV infection-associated disease pathogenesis (16–24). Different settings have given rise to quite distinct phenotypes, including B-cell type NHL (DLBCL, FBCL, and unspecified B-cell lymphomas), natural killer/T cell lymphoma (NKTCL), nonmalignant lymphoproliferative disorder (LPD), extremely rare HL, HLH, and arthritis (16–24). Despite considerable efforts (16–24), EBVaHL has not been properly produced in the humanized mouse setting model, owing to inappropriate animal models and a lack of in-depth analyses. After an initial report of infected humanized mice, HRS-like cells appeared to be extremely rare in the spleens of infected humanized mice; however, the findings were inconclusive (18). Here we report direct evidence of EBVaHL or HL-like neoplasms in multiple humanized mice in which T cells were predominant over B cells. Our study demonstrates that EBV-infected humanized mice display additional EBV-associated pathogenesis, including DLBCL and hemophagocytic lymphohistiocytosis (16, 17).     

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.     

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.     

Hemolysis-induced lethality involves inflammasome activation by heme

The increase of extracellular heme is a hallmark of hemolysis or extensive cell damage. Heme has prooxidant, cytotoxic, and inflammatory effects, playing a central role in the pathogenesis of malaria, sepsis, and sickle cell disease. However, the mechanisms by which heme is sensed by innate immune cells contributing to these diseases are not fully characterized. We found that heme, but not porphyrins without iron, activated LPS-primed macrophages promoting the processing of IL-1β dependent on nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). The activation of NLRP3 by heme required spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K⁺ efflux, whereas it was independent of heme internalization, lysosomal damage, ATP release, the purinergic receptor P2X7, and cell death. Importantly, our results indicated the participation of macrophages, NLRP3 inflammasome components, and IL-1R in the lethality caused by sterile hemolysis. Thus, understanding the molecular pathways affected by heme in innate immune cells might prove useful to identify new therapeutic targets for diseases that have heme release.Hemolysis, hemorrhage, and rhabdomyolysis cause the release of large amounts of hemoproteins to the extracellular space, which, once oxidized, release the heme moiety, a potentially harmful molecule due to its prooxidant, cytotoxic, and inflammatory effects (1, 2). Scavenging proteins such as haptoglobin and hemopexin bind hemoglobin and heme, respectively, promoting their clearance from the circulation and delivery to cells involved with heme catabolism. Heme oxygenase cleaves heme and generates equimolar amounts of biliverdin, carbon monoxide (CO) and iron (2). Studies using mice deficient for haptoglobin (Hp), hemopexin (Hx), and heme oxygenase 1 (HO-1) demonstrate the importance of these proteins in controlling the deleterious effects of heme. Both Hp^−/− and Hx^−/− mice have increased renal damage after acute hemolysis induced by phenyhydrazine (Phz) compared with wild-type mice (3, 4). Mice lacking both proteins present splenomegaly and liver inflammation composed of several foci with leukocyte infiltration after intravascular hemolysis (5). Hx protect mice against heme-induced endothelial damage improving liver and cardiovascular function (6–8). Lack of heme oxygenase 1 (Hmox1^−/−) causes iron overload, increased cell death, and tissue inflammation under basal conditions and upon inflammatory stimuli (9–15). This salutary effect of HO-1 has been attributed to its effect of reducing heme amounts as well as generating the cytoprotective molecules, biliverdin and CO.Heme induces neutrophil migration in vivo and in vitro (16, 17), inhibits neutrophil apoptosis (18), triggers cytokine and lipid mediator production by macrophages (19, 20), and increases the expression of adhesion molecules and tissue factor on endothelial cells (21–23). Heme cooperates with TNF, causing hepatocyte apoptosis in a mechanism dependent on reactive oxygen species (ROS) generation (12). Whereas heme-induced TNF production depends on functional toll-like receptor 4 (TLR4), ROS generation in response to heme is TLR4 independent (19). We recently observed that heme triggers receptor-interacting protein (RIP)1/3-dependent macrophage-programmed necrosis through the induction of TNF and ROS (15). The highly unstable nature of iron is considered critical for the ability of heme to generate ROS and to cause inflammation. ROS generated by heme has been mainly attributed to the Fenton reaction. However, recent studies suggest that heme can generate ROS through multiple sources, including NADPH oxidase and mitochondria (22, 24–27).Heme causes inflammation in sterile and infectious conditions, contributing to the pathogenesis of hemolytic diseases, subarachnoid hemorrhage, malaria, and sepsis (11, 13, 24, 28), but the mechanisms by which heme operates in different conditions are not completely understood. Blocking the prooxidant effects of heme protects cells from death and prevents tissue damage and lethality in models of malaria and sepsis (12, 13, 15). Importantly, two recent studies demonstrated the pathogenic role of heme-induced TLR4 activation in a mouse model of sickle cell disease (29, 30). These results highlight the great potential of understanding the molecular mechanisms of heme-induced inflammation and cell death as a way to identify new therapeutic targets.Hemolysis and heme synergize with microbial molecules for the induction of inflammatory cytokine production and inflammation in a mechanism dependent on ROS and Syk (24). Processing of pro–IL-1β is dependent on caspase-1 activity, requiring assembly of the inflammasome, a cytosolic multiprotein complex composed of a NOD-like receptor, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 (31–33). The most extensively studied inflammasome is the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). NLRP3 and pro–IL-1β expression are increased in innate immune cells primed with NF-κB inducers such as TLR agonists and TNF (34, 35). NLRP3 inflammasome is activated by several structurally nonrelated stimuli, such as endogenous and microbial molecules, pore-forming toxins, and particulate matter (34, 35). The activation of NLRP3 involves K⁺ efflux, increase of ROS and Syk phosphorylation. Importantly, critical roles of NLRP3 have been demonstrated in a vast number of diseases (34, 36). We hypothesize that heme causes the activation of the inflammasome and secretion of IL-1β. Here we found that heme triggered the processing and secretion of IL-1β dependently on NLRP3 inflammasome in vitro and in vivo. The activation of NLRP3 by heme was dependent on Syk, ROS, and K⁺ efflux, but independent of lysosomal leakage, ATP release, or cell death. Finally, our results indicated the critical role of macrophages, the NLRP3 inflammasome, and IL-1R to the lethality caused by sterile hemolysis.     

Programmed cell death 1 inhibits inflammatory helper T-cell development through controlling the innate immune response

Programmed cell death 1 (PD-1) is an inhibitory coreceptor on immune cells and is essential for self-tolerance because mice genetically lacking PD-1 (PD-1^−/−) develop spontaneous autoimmune diseases. PD-1^−/− mice are also susceptible to severe experimental autoimmune encephalomyelitis (EAE), characterized by a massive production of effector/memory T cells against myelin autoantigen, the mechanism of which is not fully understood. We found that an increased primary response of PD-1^−/− mice to heat-killed mycobacteria (HKMTB), an adjuvant for EAE, contributed to the enhanced production of T-helper 17 (Th17) cells. Splenocytes from HKMTB-immunized, lymphocyte-deficient PD-1^−/− recombination activating gene (RAG)2^−/− mice were found to drive antigen-specific Th17 cell differentiation more efficiently than splenocytes from HKMTB-immunized PD-1^+/+ RAG2^−/− mice. This result suggested PD-1’s involvement in the regulation of innate immune responses. Mice reconstituted with PD-1^−/− RAG2^−/− bone marrow and PD-1^+/+ CD4⁺ T cells developed more severe EAE compared with the ones reconstituted with PD-1^+/+ RAG2^−/− bone marrow and PD-1^+/+ CD4⁺ T cells. We found that upon recognition of HKMTB, CD11b⁺ macrophages from PD-1^−/− mice produced very high levels of IL-6, which helped promote naive CD4⁺ T-cell differentiation into IL-17–producing cells. We propose a model in which PD-1 negatively regulates antimycobacterial responses by suppressing innate immune cells, which in turn prevents autoreactive T-cell priming and differentiation to inflammatory effector T cells.Autoimmune disease development is impacted by both genetic and environmental factors. Programmed cell death 1 (PD-1) is a type I membrane protein that delivers inhibitory signals to immune cells upon the binding of its ligand, PD-L1 or PD-L2 (1). PD-1 has been shown to be important for self-tolerance because spontaneous autoimmune diseases develop in PD-1^−/− mice (2–4). A single-nucleotide polymorphism that affects PD-1 expression is associated with autoimmune diseases in humans, such as systemic lupus erythematosus (5), type I diabetes (6), rheumatoid arthritis (7), and multiple sclerosis (MS) (8), suggesting that PD-1 deficiency may be a genetic factor involved in the development of autoimmunity.Experimental autoimmune encephalomyelitis (EAE) is a rodent model of T-cell–mediated inflammatory disease in the central nervous system (CNS), causing demyelination, axonal damage, and paralysis, and is a commonly used model for human MS. Previous reports suggested that PD-1 functions to attenuate EAE. PD-1 and its ligands were found to be strongly expressed on immune infiltrates in the CNS during the peak phase of EAE (9–11). In EAE studies, PD-1–deficient mice or the use of blocking antibodies that inhibit PD-1 engagement by ligands resulted in earlier disease onset, increased inflammatory infiltrates, and increased severity of clinical symptoms compared with normal disease progression (10–16). It has been demonstrated that ligand engagement of PD-1 inhibits T-cell activation, expansion, and cytokine production (17–19). Similarly, in EAE, PD-1 signaling in CNS-specific helper T cells may inhibit their expansion and secretion of inflammatory cytokines (10–12). Recently, T-helper 17 (Th17) cells were shown to be involved in EAE by producing IL-17 and GM-CSF (20, 21). Two reports showed that PD-1^−/− mice mount an augmented Th17 response to EAE induction (14, 16). However, the fundamental mechanisms by which PD-1 regulates antigen-specific Th17 cell differentiation, expansion, and effector function in EAE remain to be understood.To induce EAE, mice are immunized with myelin autoantigens in an emulsion of Mycobacterium tuberculosis (MTB)-derived adjuvants, causing a strong innate inflammatory response, leading to Th skewing (22). Curiously, recent studies showed that PD-1^−/− mice exhibited an altered response to infection with mycobacteria, characterized by uncontrolled bacterial burden; massive production of cytokines, termed “cytokine storm”; and early death (23–25). We wondered if this unique response of PD-1^−/− mice to mycobacteria contributed to their Th response in EAE.In this study, we took a combination of genetic and immunological approaches in which the innate response to MTB-derived adjuvant and antigen-specific T-cell polarization were separately analyzed. The present data suggest that an enhanced innate response of PD-1^−/− mice to MTB contributes to the susceptibility of these mice to severe EAE. We propose a previously undescribed function of PD-1 in controlling the basal state of the innate immune response, the failure of which can cause the activation of adaptive immune responses, provoking autoimmunity.