Toll-like Receptor Recognition Research Articles

Card9 and MyD88 differentially regulate Th17 immunity to the commensal yeast Malassezia in the murine skin.

The fungal community of the skin microbiome is dominated by a single genus, Malassezia. Besides its symbiotic lifestyle at the host interface, this commensal yeast has also been associated with diverse inflammatory skin diseases in humans and pet animals. Stable colonization is maintained by antifungal type 17 immunity. The mechanisms driving Th17 responses to Malassezia remain, however, unclear. Here, we show that the C-type lectin receptors Mincle, Dectin-1, and Dectin-2 recognize conserved patterns in the cell wall of Malassezia and induce dendritic cell activation in vitro, while only Dectin-2 is required for Th17 activation during experimental skin colonization in vivo. In contrast, Toll-like receptor recognition was redundant in this context. Instead, inflammatory IL-1 family cytokines signaling via MyD88 were also implicated in Th17 activation in a T cell-intrinsic manner. Taken together, we characterized the pathways contributing to protective immunity against the most abundant member of the skin mycobiome. This knowledge contributes to the understanding of barrier immunity and its regulation by commensals and is relevant considering how aberrant immune responses are associated with severe skin pathologies.

Interactions between toll-like receptors signaling pathway and gut microbiota in host homeostasis.

Toll-like receptors (TLRs) are a family of fundamental pattern recognition receptorsin the innate immune system, constituting the first line of defense against endogenous and exogenous antigens. The gut microbiota, a collection of commensal microorganisms in the intestine, is a major source of exogenous antigens. The components and metabolites of the gut microbiota interact with specific TLRs to contribute to whole-body immune and metabolic homeostasis. This review aims to summarize the interaction between the gut microbiota and TLRsignaling pathways and to enumerate the role of microbiota dysbiosis-induced TLR signaling pathways in obesity, inflammatory bowel disease (IBD), and colorectal cancer (CRC). Through the recognition of TLRs, the microbiota facilitates the development of both the innate and adaptive immune systems, while the immune system monitors dynamic changes in the commensal bacteria to maintain the balance of the host-microorganism symbiosis. Dysbiosis of the gut microbiota can induce a cascade of inflammatory and metabolic responses mediated by TLRsignaling pathways, potentially resulting in various metabolic and inflammatory diseases. Understanding the crosstalk between TLRs and the gut microbiota contributes to potential therapeutic applications in related diseases, offering new avenues for treatment strategies in conditions like obesity, IBD,and CRC.

Placental TLR recognition of salivary and subgingival microbiota is associated with pregnancy complications

BackgroundPre-term birth, the leading cause of neonatal mortality, has been associated with maternal periodontal disease and the presence of oral pathogens in the placenta. However, the mechanisms that underpin this link are not known. This investigation aimed to identify the origins of placental microbiota and to interrogate the association between parturition complications and immune recognition of placental microbial motifs.61FbVQciH1C2BbwKYgvy28Video MethodsSaliva, plaque, serum, and placenta were collected during 130 full-term (FT), pre-term (PT), or pre-term complicated by pre-eclampsia (PTPE) deliveries and subjected to whole-genome shotgun sequencing. Real-time quantitative PCR was used to measure toll-like receptors (TLR) 1–10 expression in placental samples. Source tracking was employed to trace the origins of the placental microbiota.ResultsWe discovered 10,007 functionally annotated genes representing 420 taxa in the placenta that could not be attributed to contamination. Placental microbial composition was the biggest discriminator of pregnancy complications, outweighing hypertension, BMI, smoking, and maternal age. A machine-learning algorithm trained on this microbial dataset predicted PTPE and PT with error rates of 4.05% and 8.6% (taxonomy) and 6.21% and 7.38% (function). Logistic regression revealed 32% higher odds of parturition complication (95% CI 2.8%, 81%) for every IQR increase in the Shannon diversity index after adjusting for maternal smoking status, maternal age, and gravida. We also discovered distinct expression patterns of TLRs that detect RNA- and DNA-containing antigens in the three groups, with significant upregulation of TLR9, and concomitant downregulation of TLR7 in PTPE and PT groups, and dense correlation networks between microbial genes and these TLRs. 70–82% of placental microbiota were traced to serum and thence to the salivary and subgingival microbiomes. The oral and serum microbiomes of PTPE and PT groups displayed significant enrichment of genes encoding iron transport, exosome, adhesion, quorum sensing, lipopolysaccharide, biofilm, and steroid degradation.ConclusionsWithin the limits of cross-sectional analysis, we find evidence to suggest that oral bacteria might translocate to the placenta via serum and trigger immune signaling pathways capable of inducing placental vascular pathology. This might explain, in part, the higher incidence of obstetric syndromes in women with periodontal disease.

Masking of typical TLR4 and TLR5 ligands modulates inflammation and resolution by Helicobacter pylori.

Helicobacter pylori is a paradigm of chronic bacterial infection and is associated with peptic ulceration and malignancies. H. pylori uses specific masking mechanisms to avoid canonical ligands from activating Toll-like receptors (TLRs), such as lipopolysaccharide (LPS) modification and specific flagellin sequences that are not detected by TLR4 and TLR5, respectively. Thus, it was believed for a long time that H. pylori evades TLR recognition as a crucial strategy for immune escape and bacterial persistence. However, recent data indicate that multiple TLRs are activated by H. pylori and play a role in the pathology. Remarkably, H. pylori LPS, modified through changes in acylation and phosphorylation, is mainly sensed by other TLRs (TLR2 and TLR10) and induces both pro- and anti-inflammatory responses. In addition, two structural components of the cag pathogenicity island-encoded type IV secretion system (T4SS), CagL and CagY, were shown to contain TLR5-activating domains. These domains stimulate TLR5 and enhance immunity, while LPS-driven TLR10 signaling predominantly activates anti-inflammatory reactions. Here, we discuss the specific roles of these TLRs and masking mechanisms during infection. Masking of typical TLR ligands combined with evolutionary shifting to other TLRs is unique for H. pylori and has not yet been described for any other species in the bacterial kingdom. Finally, we highlight the unmasked T4SS-driven activation of TLR9 by H. pylori, which mainly triggers anti-inflammatory responses.

Evasion of toll-like receptor recognition by Escherichia coli is mediated via population level regulation of flagellin production.

Uropathogenic Escherichia coli is a major cause of urinary tract infections. Analysis of the innate immune response in immortalized urothelial cells suggests that the bacterial flagellar subunit, flagellin, is key in inducing host defenses. A panel of 48 clinical uro-associated E. coli isolates recovered from either cystitis, pyelonephritis asymptomatic bacteriuria (ABU) or UTI-associated bacteraemia infections were characterized for motility and their ability to induce an innate response in urothelial cells stably transfected with a NF-κB luciferase reporter. Thirty-two isolates (67%) were identified as motile with strains recovered from cystitis patients exhibiting an uneven motility distribution pattern; seven of the cystitis isolates were associated with a > 5-fold increase in NF-κB signaling. To explore whether the NF-κB signaling response reflected antigenic variation, flagellin was purified from 14 different isolates. Purified flagellin filaments generated comparable NF-κB signaling responses, irrespective of either the source of the isolate or H-serotype. These data argued against any variability between isolates being related to flagellin itself. Investigations also argued that neither TLR4 dependent recognition of bacterial lipopolysaccharide nor growth fitness of the isolates played key roles in leading to the variable host response. To determine the roles, if any, of flagellar abundance in inducing these variable responses, flagellar hook numbers of a range of cystitis and ABU isolates were quantified. Images suggested that up to 60% of the isolate population exhibited flagella with the numbers averaging between 1 and 2 flagella per bacterial cell. These data suggest that selective pressures exist in the urinary tract that allow uro-associated E. coli strains to maintain motility, but exploit population heterogeneity, which together function to prevent host TLR5 recognition and bacterial killing.

Scratching the Surface Takes a Toll: Immune Recognition of Viral Proteins by Surface Toll-like Receptors.

Early innate viral recognition by the host is critical for the rapid response and subsequent clearance of an infection. Innate immune cells patrol sites of infection to detect and respond to invading microorganisms including viruses. Surface Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) that can be activated by viruses even before the host cell becomes infected. However, the early activation of surface TLRs by viruses can lead to viral clearance by the host or promote pathogenesis. Thus, a plethora of research has attempted to identify specific viral ligands that bind to surface TLRs and mediate progression of viral infection. Herein, we will discuss the past two decades of research that have identified specific viral proteins recognized by cell surface-associated TLRs, how these viral proteins and host surface TLR interactions affect the host inflammatory response and outcome of infection, and address why controversy remains regarding host surface TLR recognition of viral proteins.

Nanoparticle-based delivery strategies of multifaceted immunomodulatory RNA for cancer immunotherapy

Although cancer immunotherapy has emerged as a novel cancer treatment modality, it still suffers from low therapeutic efficacy in clinics due to the presence of a low number of activated immune cells and immunosuppressive factors in the tumor microenvironment (TME). Immunomodulatory ribonucleic acids (RNAs) have been developed to improve the therapeutic efficacy of cancer immunotherapy through either regulating target cell functions [i.e., messenger RNA (mRNA) or small interfering RNA (siRNA)] or stimulating immune cells [i.e., toll-like receptors (TLRs) or cytosolic retinoic acid-inducible gene I (RIG-I) agonist]. However, RNA-based therapeutics face many biological barriers, including ineffective delivery to target cells, degradation by ribonucleases (RNases), and difficulties in passing through the cellular membranes. In this review, we discuss nanoparticle-based delivery strategies that can overcome these hurdles to enhance RNA-based immunomodulation in cancer immunotherapy. Various nanoparticle-based delivery has been reported to increase the delivery efficacy of RNAs, by improving cellular uptake, RNA stability, and accumulation at the desired sites (target cells and intracellular compartments). The nanoparticle-based delivery of multifaceted immunomodulatory RNAs could enhance cancer immunotherapy through the regulating functions of immune cells, tumor cells, and immunosuppressive factors as well as stimulating the immune cells by recognition of endosomal TLRs and cytosolic RIG-I. Nanotechnology-assisted RNA-based therapeutics are expected to offer tremendous potential and advances for treating cancer, viral infections, and other diseases.

Research progress on Toll-like receptor signal transduction and its roles in antimicrobial immune responses.

When microorganisms invade a host, the innate immune system first recognizes the pathogen-associated molecular patterns of these microorganisms through pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are known transmembrane PRRs existing in both invertebrates and vertebrates. Upon ligand recognition, TLRs initiate a cascade of signaling events; promote the pro-inflammatory cytokine, type I interferon, and chemokine expression; and play an essential role in the modulation of the host’s innate and adaptive immunity. Therefore, it is of great significance to improve our understanding of antimicrobial immune responses by studying the role of TLRs and their signal molecules in the host’s defense against invading microbes. This paper aims to summarize the specificity of TLRs in recognition of conserved microbial components, such as lipoprotein, lipopolysaccharide, flagella, endosomal nucleic acids, and other bioactive metabolites derived from microbes. This set of interactions helps to elucidate the immunomodulatory effect of TLRs and the signal transduction changes involved in the infectious process and provide a novel therapeutic strategy to combat microbial infections.

Selective targeting of the TLR4 co-receptor, MD2, prevents colon cancer growth and lung metastasis.

Toll-like receptor (TLR) signaling is an emerging pathway in tumor cell invasion and metastasis. Myeloid differentiation protein-2 (MD2) contributes to ligand recognition and activation of TLRs in response to exogenous microbial insults or endogenous agents. We hypothesized that blocking MD2 using a specific inhibitor would prevent TLR4-mediated inflammatory responses and metastatic cancer growth. Here, we report that a MD2 inhibitor, L6H21, inhibited migration and invasion of LPS-activated colon cancer CT26.WT cells. These activities were accompanied by inhibition of nuclear factor-κB (NF-κB) activation, and thereby inhibition of the production of pro-inflammatory cytokines and adhesive molecules in colon cancer cells. Furthermore, L6H21 inhibited CT26.WT metastasis to the lung in BALB/c mice as well as suppressed colitis-induced colon cancer induced by azoxymethane/dextran sulfate sodium (AOM/DSS). Taken together, our results demonstrated that L6H21 suppressed tumor invasion and metastasis through blocking TLR4-MD2/NF-κB signaling axis. These findings reveal that inhibition of MD2 may be an important target for the development of colon cancer therapies.

HP1717 Contributes to Streptococcus suis Virulence by Inducing an Excessive Inflammatory Response and Influencing the Biosynthesis of the Capsule.

Streptococcus suis 2 (SS2) is an important zoonotic pathogen that substantially harms the swine industry and poses threats to human health. Excessive inflammation is considered to be a hallmark of SS2 infection because it is responsible for most clinical signs of SS2, especially streptococcal toxic shock-like syndrome. However, the current knowledge of SS2-induced excessive inflammation remains limited. In this study, we identified HP1717 as a novel extracellular pro-inflammatory protein in SS2 that can induce robust expression of inflammatory cytokines in RAW264.7 macrophages. Notably, the pro-inflammatory ability of HP1717 was dose-dependent and heat-sensitive, and it required the recognition of Toll-like receptor 2 (TLR2) and the phosphorylation of both extracellular signal-regulated kinases 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Further, by constructing a deletion mutant, we demonstrated that HP1717 significantly influenced the biosynthesis of the bacterial capsule, which plays a critical role in the virulence of SS2 by interfering with the ability of host immune cells to phagocytize and kill the pathogen. Indeed, the mutant strain displayed reduced resistance to whole-blood killing compared with the wild strain. Finally, murine experiments indicated that the deletion of hp1717 in SS2 reduced the lethality, pro-inflammatory activity, and bacterial loads in mice. Collectively, our data reveal HP1717 as a novel virulence-related factor of SS2 that can induce an excessive inflammatory response and significantly affect the bacterial capsule, thus expanding our understanding of the pathogenesis of S. suis.

Putative link between Polo-like kinases (PLKs) and Toll-like receptor (TLR) signaling in transformed and primary human immune cells

Toll-like receptors (TLRs) are important sentinels of bacterial and viral infection and thus fulfil a critical sensory role in innate immunity. Polo-like kinases (PLKs), a five membered family of Ser/Thr protein kinases, have long been studied for their role in mitosis and thus represent attractive therapeutic targets in cancer therapy. Recently, PLKs were implicated in TLR signaling in mice but the role of PLKs in TLR signaling in untransformed primary immune cells has not been addressed, even though PLK inhibitors are in clinical trials. We here identified several phospho-serine and phospho-threonine residues in the known TLR pathway kinases, Interleukin-1 receptor-associated kinase (IRAK) 2 and IRAK4. These sites lie in canonical polo-box motifs (PBM), sequence motifs known to direct recruitment of PLKs to client proteins. Interestingly, PLK1 was phosphorylated and PLK 2 and 3 mRNA induced upon TLR stimulation in primary immune cells, respectively. In whole blood, PLK inhibition disparately affected TLR mediated cytokine responses in a donor- and inhibitor-dependent fashion. Collectively, PLKs may thus potentially interface with TLR signaling in humans. We propose that temporary PLK inhibitor-mediated blockade of TLR-signaling in certain patients receiving such inhibitors during cancer treatment may cause adverse effects such as an increased risk of infections due to a then compromised ability of the TLR recognition system to sense and initiate cytokine responses to invading microbes.

Human cathelicidin improves colonic epithelial defenses against Salmonella typhimurium by modulating bacterial invasion, TLR4 and pro-inflammatory cytokines.

The intestinal mucosa contributes to frontline gut defenses by forming a barrier (physical and biochemical) and preventing the entry of pathogenic microbes. One innate role of the human colonic epithelium is to secrete cathelicidin, a peptide with broad antimicrobial and immunomodulatory functions. In this study, the effect of cathelicidin in the maintenance of epithelial integrity, Toll-like receptor recognition, bacterial invasion and initiation of inflammatory response against Salmonella typhimurium is investigated in cultured human colonic epithelium. We found exogenous human cathelicidin restores the epithelial integrity in S. typhimurium-infected colonic epithelial (T84) cells by mostly post-translational effects associated with reorganization of zonula occludens (ZO)-1 tight junction proteins. Endogenous cathelicidin prevents S. typhimurium internalization as shown in colonic epithelial cells genetically deficient in the only human cathelicidin, LL-37 (shLL-37). Moreover, supplementation of shLL-37 cells with synthetic LL-37 reduces the grade of S. typhimurium internalization in a dose-dependent manner. Mechanistically, shLL-37 cells have lower gene expression of TLR4 and pro-inflammatory cytokine IL-1β in response to S. typhimurium. Thus, human cathelicidin aids in the early colonic epithelial defenses against enteric S. typhimurium by preventing bacterial invasion and maintaining epithelial barrier integrity, likely to occur due to the production of sensing TLR4 and pro-inflammatory cytokines.

Tuned Cationic Dendronized Polymer: Molecular Scavenger for Rheumatoid Arthritis Treatment

AbstractCell‐free deoxyribonucleic acid (cfDNA) released from either dead or damaged cells serves as a key autoantigen in rheumatoid arthritis (RA). They can be recognized by nucleic acid (NA) sensors such as the toll‐like receptor (TLR), leading to activation of the innate immune system and chronic inflammation. Developed here is a cationic molecular scavenger, by screening cationic dendronized polymers, which eliminates cfDNA and inhibits TLR recognition and nucleic‐acid‐induced inflammation. The structure–property study demonstrates that toxicity, NA binding capacity, and biodistribution could be balanced to achieve maximum therapeutic effect by exquisite control of the molecular structure. In addition, the optimized cationic polymer effectively inhibited joint swelling, synovial hyperplasia, and bone destruction in collagen‐induced arthritis (CIA) rat models. The results offer support for synthetic polymers offering new paradigm in autoimmune disease treatment.

Tuned Cationic Dendronized Polymer: Molecular Scavenger for Rheumatoid Arthritis Treatment

Cell-free deoxyribonucleic acid (cfDNA) released from either dead or damaged cells serves as a key autoantigen in rheumatoid arthritis (RA). They can be recognized by nucleic acid (NA) sensors such as the toll-like receptor (TLR), leading to activation of the innate immune system and chronic inflammation. Developed here is a cationic molecular scavenger, by screening cationic dendronized polymers, which eliminates cfDNA and inhibits TLR recognition and nucleic-acid-induced inflammation. The structure-property study demonstrates that toxicity, NA binding capacity, and biodistribution could be balanced to achieve maximum therapeutic effect by exquisite control of the molecular structure. In addition, the optimized cationic polymer effectively inhibited joint swelling, synovial hyperplasia, and bone destruction in collagen-induced arthritis (CIA) rat models. The results offer support for synthetic polymers offering new paradigm in autoimmune disease treatment.

Integrin β3 interacts with Toll-like receptor 4 to up-regulate CD14 expression in macrophages and contributes to sepsis induced acute lung injury

Sepsis is the major challenge in clinical practice with high mortality.1 Toll-like receptors (TLRs) play a fundamental role in sepsis. CD14 is a co-receptor of many Toll-like receptors.2 In our previous study, we found integrin β3 deficiency decreased sepsis induced CD14 expression and attenuated acute lung injury. In this study we used a cecal ligation and puncture (CLP) induced sepsis model and LPS-treated macrophages to uncover the mechanism of integrin β3 modulated CD14 expression under inflammatory conditions. In vivo studies. To define the role of integrin β3 in macrophages in a septic mouse, macrophages were derived from bone marrow and retransfusion, with or without prior depletion of macrophages by clodronate-containing liposomes. To detect morphology changes, lungs were obtained at 24 h after CLP and fixed with 4% paraformaldehyde (PFA) for haematoxylin–eosin (HE) stain. To detect the inflammatory response, bronchoalveolar lavage fluid (BALF) was obtained and tested for the levels of tumour necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-β via enzyme-linked immunosorbent assay (ELISA). To detect CD14 expression tissue, the lung was homogenised and lysed for western blot. In vitro studies. To further investigate if integrin β3 is directly or indirectly involved in CD14 expression, we used (1) wild type macrophages pretreated with integrin β3 inhibitor P11 or anti-antibody or (2) integrin β3–/– macrophages stimulated with LPS for different times and measured the levels of TNF-α and IL-6 via ELISA and CD14 via western blot and flow cytometry. Secondly, integrin β3–/– macrophages treated with recombinant CD14 protein and LPS for different time points were used and TNF-α and IL-6 secretion was measured by ELISA. Thirdly, macrophages were isolated from wild type mice and pretreated with polymyxin B (PMB) or TAK-242 or isolated from C3He/J mice and stimulated with LPS for 24 h to detect CD14 expression via western blot or flow cytometry. Lastly, co-transfected 293T cells with integrin β3-HA and TLR4-Flag plasmids were then stimulated with LPS for 30 or 60 min, and the interaction by co-immunoprecipitation was assessed. In wild type mice with prior depletion of macrophages followed by retransfusion of wild type macrophages, lung injury, inflammatory cytokines, and CD14 expression was significantly increased compared with retransfusion of integrin β3 deficient macrophages. In integrin β3 deficient mice with prior depletion of macrophages and retransfusion of wild type macrophages, the lung injury, inflammatory cytokines, and CD14 expression was increased compared with retransfusion of integrin β3 deficient macrophages. Blocking integrin β3 pathway by P11 or anti-antibody inhibited LPS-induced TNF-α and IL-6 release at early but not late times, but had no effect on CD14 expression in wild type macrophages. In integrin β3 deficient macrophages, LPS-induced TNF-α and IL-6 release was significantly decreased from 4 to 24 h as well as CD14 expression. Recombination mCD14 reversed integrin β3 deficiency decreased TNF-α and IL-6 release. Blocking LPS by PMB or inhibition of LPS–Toll-like receptor 4 (TLR4) pathway by TAK-242 or TLR4 mutant macrophages, LPS-induced CD14 expression was abolished. Co-IP results showed that TLR4 and integrin β3 (slightly) interacted during the resting state, and the interaction was increased after LPS stimulation (see Fig. 1). Integrin β3 is involved in TLR activation-induced CD14 up-regulation and contributed to sepsis-induced acute lung injury. Deletion of integrin β3 in macrophages not only inhibited integrin β3 pathway activation-induced inflammatory cytokine release but also decreased TLR4 activation-induced CD14 up-regulation and reduced CD14 effect on TLR recognition and enhanced inflammation. Specifically targeting integrin β3/TLR4-CD14 signalling may be a potential treatment strategy in polymicrobial sepsis.

Targeting and Recognition of Toll-Like Receptors by Plant and Pathogen Lectins.

We have reported that some lectins act as agonists of toll-like receptors (TLRs) and have immunomodulatory properties. The plant lectin ArtinM, for example, interacts with N-glycans of TLR2, whereas other lectins of microbial origin interact with TLR2 and TLR4. Expression of the receptors on the surface of antigen-presenting cells exposes N-glycans that may be targeted by lectins of different structures, specificities, and origins. In vitro, these interactions trigger cell signaling that leads to NF-κB activation and production of the Th1 polarizing cytokine IL-12. In vivo, a same sequence of events follows the administration of an active lectin to mice infected with an intracellular pathogen, conferring resistance to the pathogen. The lectins of the human pathogens Toxoplasma gondii (TgMIC1 and TgMIC4) and Paracoccidioides brasiliensis (Paracoccin), by recognition and activation of TLR2 and TLR4, induce cell events and in vivo effects comparable to the promoted by the plant lectin ArtinM. In this article, we highlight these two distinct mechanisms for activating antigen-presenting cells. On the one hand, TLRs act as sensors for the presence of conventional pathogen-associated molecular patterns, such as microbial lipids. On the other hand, we showed that TLR-mediated cell activation might be triggered by an alternative way, in which lectins bind to TLRs N-glycans and stimulate cells to increase the expression of pro-inflammatory cytokines. This process may lead to the development of new pharmaceutical tools that promote protective immune responses directed against intracellular pathogens and tumors.

HP1330 Contributes to Streptococcus suis Virulence by Inducing Toll-Like Receptor 2- and ERK1/2-Dependent Pro-inflammatory Responses and Influencing In Vivo S. suis Loads.

Streptococcus suis 2 (SS2) has evolved into a highly invasive pathogen responsible for two large-scale outbreaks of streptococcal toxic shock-like syndrome (STSLS) in China. Excessive inflammation stimulated by SS2 is considered a hallmark of STSLS, even it also plays important roles in other clinical symptoms of SS2-related disease, including meningitis, septicemia, and sudden death. However, the mechanism of SS2-caused excessive inflammation remains poorly understood. Here, a novel pro-inflammatory protein was identified (HP1330), which could induce robust expression of pro-inflammatory cytokines (TNF-α, MCP-1, and IL-1β) in RAW264.7 macrophages. To evaluate the role of HP1330 in SS2 virulence, an hp1330-deletion mutant (Δhp1330) was constructed. In vitro, hp1330 disruption led to a decreased pro-inflammatory ability of SS2 in RAW 264.7 macrophages. In vivo, Δhp1330 showed reduced lethality, pro-inflammatory activity, and bacterial loads in mice. To further elucidate the mechanism of HP1330-induced pro-inflammatory cytokine production, antibody blocking and gene-deletion experiments with macrophages were performed. The results revealed that the pro-inflammatory activity of HP1330 depended on the recognition of toll-like receptor 2 (TLR2). Furthermore, a specific inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathways could significantly decrease HP1330-induced pro-inflammatory cytokine production, and western blot analysis showed that HP1330 could induce activation of the ERK1/2 pathway. Taken together, our findings demonstrate that HP1330 contributes to SS2 virulence by inducing TLR2- and ERK1/2-dependent pro-inflammatory cytokine production and influencing in vivo bacterial loads, implying that HP1330 may be associated with STSLS caused by SS2.

Toward a structural understanding of nucleic acid-sensing Toll-like receptors in the innate immune system.

The history of mankind has been plagued by the tug of war with viral infections. Toll-like receptors (TLRs) and other receptors of the innate immune system constitute an early defense system against invading viruses by recognizing the viral genetic material, the nucleic acids (NAs). Agonistic ligands of NA-sensing TLRs play an emerging role in the treatment of viral diseases, demonstrating a crucial role of these receptors. Recently, crystal structures have afforded new insights into TLR recognition of NAs. An aberrant activation by self-NAs, which leads to the inflammation and autoimmunity, is avoided by strict regulation of NA-TLR interaction at multiple check-points. This Review summarizes the novel structural understanding of NA-sensing by TLRs and regulatory mechanisms of these receptors.

Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry.

Many enveloped viruses enter cells through endocytosis. Viral spike proteins drive the fusion of viral and endosomal membranes to facilitate insertion of the viral genome into the cytoplasm. Human coronavirus 229E (HCoV-229E) utilizes endosomal cathepsin L to activate the spike protein after receptor binding. Here, we found that clinical isolates of HCoV-229E preferentially utilize the cell surface protease TMPRSS2 rather than endosomal cathepsin L. The endosome is a main site of Toll-like receptor recognition, which then triggers an innate immune response; therefore, HCoV-229E presumably evolved to bypass the endosome by entering the cell via TMPRSS2. Thus, the virus uses a simple mechanism to evade the host innate immune system. Therefore, therapeutic agents for coronavirus-mediated diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), should target cell surface TMPRSS2 rather than endosomal cathepsin.

Source of CpG Depletion in the HIV-1 Genome.

The dinucleotide CpG is highly underrepresented in the genome of human immunodeficiency virus type 1 (HIV-1). To identify the source of CpG depletion in the HIV-1 genome, we investigated two biological mechanisms: (1) CpG methylation-induced transcriptional silencing and (2) CpG recognition by Toll-like receptors (TLRs). We hypothesized that HIV-1 has been under selective evolutionary pressure by these mechanisms leading to the reduction of CpG in its genome. A CpG depleted genome would enable HIV-1 to avoid methylation-induced transcriptional silencing and/or to avoid recognition by TLRs that identify foreign CpG sequences. We investigated these two hypotheses by determining the sequence context dependency of CpG depletion and comparing it with that of CpG methylation and TLR recognition. We found that in both human and HIV-1 genomes the CpG motifs flanked by T/A were depleted most and those flanked by C/G were depleted least. Similarly, our analyses of human methylome data revealed that the CpG motifs flanked by T/A were methylated most and those flanked by C/G were methylated least. Given that a similar CpG depletion pattern was observed for the human genome within which CpGs are not likely to be recognized by TLRs, we argue that the main source of CpG depletion in HIV-1 is likely host-induced methylation. Analyses of CpG motifs in over 100 viruses revealed that this unique CpG representation pattern is specific to the human and simian immunodeficiency viruses.