Human Toll-like Receptor Research Articles

Prediction of deleterious non-synonymous SNPs of human TLR2 gene associated with bacterial meningitis and hearing impairment by computational approach

Background: Bacterial meningitis is the most serious infection of the central nervous system and often occurs in children under the age of 5 years and leads to hearing loss, learning disabilities. Several studies have reported the genetic variation in a gene that is involved in the innate immune system and affects the susceptibility, severity of bacterial infection during meningitis. Methodology: Hence the present study was performed to insights the single nucleotide polymorphisms in pathogen recognition genes especially those that play an essential role in the innate immune system. Toll-like receptor 2 (TLR2) was selected to analyze the association of TLR2 in bacterial meningitis. The coding and non-coding SNPs of the human TLR2 gene was retrieved from the NCBI database and were subjected to various computational prediction tools including SIFT, PolyPhen-2, Imutnat, to predict the high-risk deleterious SNPs and the effect of single amino acid substitutions and structural and functional impact of these mutations was predicted with project HOPE, SNP-GO tools. Results: The result revealed that among the 501 non-synonymous, 9 deleterious SNPs were sorted by SFIT, Polyphen-2 and I mutant serve with SFIT score, DDG score, and PISC score which are equal to 1.

Hepatitis C Virus Infection and Intrinsic Disorder in the Signaling Pathways Induced by Toll-Like Receptors.

Simple SummaryHuman hepatitis C virus causes contagious liver disease hepatitis C, which is broadly spread around the globe. Infection with this virus causes an inflammation of the liver that leads to both acute and chronic hepatitis, which, if left untreated, often results in the development of serious lifelong illnesses such as liver fibrosis, liver cirrhosis, hepatocellular carcinoma, and end-stage liver disease in humans. Toll-like receptors are a class of pattern recognition receptors that exist in liver parenchymal cells and various immune cells, and play an important role in the liver immune system by initiating multiple cellular downstream pathways in response to the recognition of pathogens such as hepatitis C virus. Proteins from the hepatitis C virus, Toll-like receptors themselves, and proteins from the pathways they activate are multifunctional. Since protein multifunctionality is commonly associated with intrinsic disorder (i.e., lack of stable 3D structures), we used an intrinsic disorder angle to examine the interplay between the hepatitis C virus infection and signaling pathways induced by Toll-like receptors. We show that almost all of these proteins contained noticeable levels of disorder, indicating that intrinsic disorder is prominently utilized in virus–host warfare.In this study, we examined the interplay between protein intrinsic disorder, hepatitis C virus (HCV) infection, and signaling pathways induced by Toll-like receptors (TLRs). To this end, 10 HCV proteins, 10 human TLRs, and 41 proteins from the TLR-induced downstream pathways were considered from the prevalence of intrinsic disorder. Mapping of the intrinsic disorder to the HCV-TLR interactome and to the TLR-based pathways of human innate immune response to the HCV infection demonstrates that substantial levels of intrinsic disorder are characteristic for proteins involved in the regulation and execution of these innate immunity pathways and in HCV-TLR interaction. Disordered regions, being commonly enriched in sites of various posttranslational modifications, may play important functional roles by promoting protein–protein interactions and support the binding of the analyzed proteins to other partners such as nucleic acids. It seems that this system represents an important illustration of the role of intrinsic disorder in virus–host warfare.

Lipid A Variants Activate Human TLR4 and the Noncanonical Inflammasome Differently and Require the Core Oligosaccharide for Inflammasome Activation.

Detection of Gram-negative bacterial lipid A by the extracellular sensor, myeloid differentiation 2 (MD2)/Toll-like receptor 4 (TLR4), or the intracellular inflammasome sensors, CASP4 and CASP5, induces robust inflammatory responses. The chemical structure of lipid A, specifically its phosphorylation and acylation state, varies across and within bacterial species, potentially allowing pathogens to evade or suppress host immunity. Currently, it is not clear how distinct alterations in the phosphorylation or acylation state of lipid A affect both human TLR4 and CASP4/5 activation. Using a panel of engineered lipooligosaccharides (LOS) derived from Yersinia pestis with defined lipid A structures that vary in their acylation or phosphorylation state, we identified that differences in phosphorylation state did not affect TLR4 or CASP4/5 activation. However, the acylation state differentially impacted TLR4 and CASP4/5 activation. Specifically, all tetra-, penta-, and hexa-acylated LOS variants examined activated CASP4/5-dependent responses, whereas TLR4 responded to penta- and hexa-acylated LOS but did not respond to tetra-acylated LOS or penta-acylated LOS lacking the secondary acyl chain at the 3' position. As expected, lipid A alone was sufficient for TLR4 activation. In contrast, both core oligosaccharide and lipid A were required for robust CASP4/5 inflammasome activation in human macrophages, whereas core oligosaccharide was not required to activate mouse macrophages expressing CASP4. Our findings show that human TLR4 and CASP4/5 detect both shared and nonoverlapping LOS/lipid A structures, which enables the innate immune system to recognize a wider range of bacterial LOS/lipid A and would thereby be expected to constrain the ability of pathogens to evade innate immune detection.

In Silico Insights on the Pro-Inflammatory Potential of Polycyclic Aromatic Hydrocarbons and the Prospective Anti-Inflammatory Capacity of Andrographis paniculata Phytocompounds.

Inflammation linked to various diseases is the biological response to certain stimuli. The pro-inflammatory potential of Polycyclic Aromatic Hydrocarbons (PAHs) as potential inducers of inflammation bound to the Toll-like Receptor 4 (TLR4) and the anti-inflammatory capacity of A. paniculata (AP) phytocompounds as prospective inhibitors of the Nuclear Factor Kappa B (NF-κB) p50 transcription factor are investigated via in silico techniques. The molecular docking of the PAHs and AP phytocompounds is performed in AutoDock Vina by calculating their binding energies. The molecular dynamics simulations (MDS) of the apo and ligand-bound complex of the top binding ligands were performed in CABS-flex. The agonists, which included the PAHs indeno(1,2,3-cd)pyrene (IP), and dibenz(a,h)anthracene (DahA), had the highest binding energies of −10 kcal/mol and −9.2 kcal/mol, respectively. The most stable antagonists in the binding site with binding energies to the NF-κB p50 were the AP phytocompounds with −5.6 kcal/mol for ergosterol peroxide and −5.3 kcal/mol for 14-deoxy-14,15-dehydroandrographolide. The MDS of the apo human TLR4 and PAH-bound TLR4, and the apo p50 and the AP phytocompound-bound NF-κB p50 showed minimal fluctuations. These results reveal that IP and DahA are significant inducers of inflammation, whereas ergosterol peroxide and 14-deoxy-14,15-dehydroandrographolide are inhibitors of the NF-κB pathway. Furthermore, the study theorizes that any inflammatory activity induced by PAH can be potentially inhibited by A. paniculata phytocompounds.

In Silico Approach in the Evaluation of Pro-Inflammatory Potential of Polycyclic Aromatic Hydrocarbons and Volatile Organic Compounds through Binding Affinity to the Human Toll-Like Receptor 4.

Polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs) are widespread across the globe, existing in the environment in complex mixtures potentially capable of initiating respiratory illnesses. Here, we use an in silico approach to evaluate the potential pro-inflammatory effects of various carcinogenic PAHs and VOCs through their binding affinity towards the human toll-like receptor 4 (TLR4). For receptors and ligands, RCSB Protein Data Bank and PubChem were used in obtaining their 3D structures, respectively. Autodock Vina was utilized to obtain the best docking poses and binding affinities of each PAH and VOC. Out of the 14 PAHs included in this study, indeno(1,2,3-cd)pyrene, benzo(ghi)perylene, and benzo[a]pyrene had the highest binding affinity values of −10, −9, and −8.9 kcal/mol, respectively. For the VOCs, out of the 10 compounds studied, benzene, 1,4-dichlorobenzene, and styrene had the highest binding affinity values of −3.6, −3.9, and −4.6 kcal/mol, respectively. Compounds with higher affinity than LPS (−4.1 kcal/com) could potentially induce inflammation, while compounds with lower affinity would be less likely to induce an inflammatory response. Meanwhile, molecular dynamics simulation and RMSF statistical analysis proved that the protein, TLR4, stably preserve its conformation despite ligand interactions. Overall, the structure of the TLR4 was considered inflexible.

Abstract 3502: AXA-042 - a novel systemic TLR2/6 agonist for anti-tumor therapy

Abstract Background: Treatment approaches that engage both the innate and adaptive immune response have the potential to transform anti-cancer therapy, especially in settings of checkpoint inhibitor insensitivity or acquired resistance. Toll-like receptors (TLRs) are a class of targets that mediate the initial cellular response to external pathogens or endogenous alarmins, activating downstream pro-inflammatory cascades and leading to the activation and recruitment of key innate subsets. TLR2 is a cell surface receptor, expressed predominantly on macrophages, dendritic cells (DC), neutrophils and subsets of NK and T cells. TLR2 signaling plays important role in NK and T cell cytolytic cell functions, as well as DC and macrophage activation. AXA-042 is a novel synthetic TLR2/6 agonist designed for systemic delivery to re-engage the innate immune response to help overcome tumour immune escape. Methods: In vitro potency and selectivity of AXA-042 was assessed across a panel of human and mouse HEK-blue TLR reporter assays. AXA-042 activity was profiled relative to other TLR agonists in human PBMC cytokine release assays. AXA-042 in vivo efficacy was evaluated in the EMT6 and CT26 syngeneic tumour models. The Nanostring nCounter™ Mouse PanCancer Immune Profiling Panel was used to identify AXA-042 tumour-localized and peripheral response signatures. Results: AXA-042 demonstrated potent and selective activity in murine and human TLR2/6 reporter assays and a differential PBMC cytokine release profile relative to other TLR agonists in vitro. Systemic delivery (QW, IV or IP) of AXA-042 significantly inhibited EMT6 and CT26 tumour growth, which was accompanied by an increase in circulating plasma levels of IL6, IL12 and CXCL10. Combination with anti-PD-1 Ab further potentiated anti-tumour response, resulting in approximately 30-40% tumour regressions. AXA-042 anti-tumour effects were accompanied by increased activation of the tumour-infiltrating myeloid cells, including DCs and macrophages, and a reduction in the M2 TAM subset. Nanostring analysis confirmed TLR and IFNγ pathway engagement and revealed an induction of gene signatures associated with macrophage, DC and NK functions. Longitudinal studies to characterize whole blood biomarkers associated with response are currently in progress. Conclusion: AXA-042 was well tolerated in vivo and demonstrated innate response engagement and anti-tumour efficacy as monotherapy and in combination with anti-PD-1 immunotherapy. AXA-042 has completed GLP toxicology studies and initiation of Phase 1/1b trial is planned for early 2022. Citation Format: Anna V. Galkin, Francesca Mercuri, Nicholas West, Ping Zhang, Weiguang Zeng, Grant McLachlan, Michael Bettess, Ian Holmes, David Jackson, Chris Smith, Christophe Demaison, Tobias Bald, Phil Kearney. AXA-042 - a novel systemic TLR2/6 agonist for anti-tumor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3502.

Abstract 242: TCTP is a target for cancer immunotherapy modulating myeloid-derived suppressor cells

Abstract Myeloid-derived suppressor cells (MDSCs) are a heterogenous group of myeloid cells that are highly suppressive to antitumor lymphocyte function and trafficking to the tumor. The accumulation of MDSCs in the tumor immune microenvironment (TIME) makes immunosuppressive TIME and progressive tumor. In fact, despite notable clinical outcomes in cancer immunotherapy, many patients are refractory or relapsed to immune checkpoint inhibitor therapy, wherein obstacles imposed by TIME contribute to a great extent. Here, we show that translationally controlled tumor protein (TCTP) released by dying tumor cells is an immunomodulator crucial to full-blown MDSC accumulation in the TIME in mouse models. We provide evidence that extracellular TCTP mediates recruitment of the polymorphonuclear MDSC (PMN-MDSC) population in the TIME, which is Toll-like receptor-2 (TLR2) dependent. For human translation, we confirmed that human TCTP binds directly to human TLR2 extracellular domain and stimulates human PBMC to produce multiple cytokines. To explore therapeutic applicability, we generated anti-TCTP antibodies for TCTP inhibition. The antibodies successfully neutralized TCTP in molecular and cellular level. The antibodies also suppressed PMN-MDSC accumulation and tumor growth. The combination of anti-TCTP antibody and immune checkpoint inhibitor showed synergistic effect on tumor growth inhibition. In conclusion, extracellular TCTP is an immunomodulator recruiting PMN-MDSCs to TIME. When TCTP is removed or neutralized, PMN-MDSC counts in TIME decreased. Anti-TCTP therapy may offer a new immunotherapy strategy. Citation Format: Jeong-Hyun Ryou, Gwanghee Lee, Tadatsugu Taniguchi, Hideyuki Yanai, Sho Hangai. TCTP is a target for cancer immunotherapy modulating myeloid-derived suppressor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 242.

Systematic review and meta-analysis of human Toll-like receptors genetic polymorphisms for susceptibility to tuberculosis infection.

Epidemiological data from the world health organization (WHO) show that Globally an estimated 10 million (range, 8.9-11.0 million) people around the world were infected with TB in 2019. M.tuberculosis (M.tb) is the major cause of tuberculosis. Infection with M.tb has varied host immune responses because of the host genetic factor and its response to the infection. Genetic polymorphism in TLRs imparts susceptibility or resistance to the host against several diseases. In the present study, a systematic review and meta-analysis were performed to describe the relationship among various TLRs and SNPs involved in M.tb infection and their association with susceptibility to pulmonary tuberculosis in various populations of the world. PubMed and Scihub databases from 2008 to 2019 were searched and 58 articles were shortlisted for the present study to explore the association between TLRs gene polymorphisms and susceptibility to tuberculosis infection. The combined analysis showed that the polymorphisms TLR1 (rs5743618), TLR1 (rs4833095), TLR2 (-196 to -174) del, TLR2 (rs3804099), TLR4 (rs4986790), TLR4 (rs4986791), TLR4 (rs7873784), TLR6 (rs5743810), TLR8 (rs3764880), TLR9 (rs5743836), TLR9 (rs352139) were significantly associated with TB disease in certain ethnic population. In our meta-analysis study, we have also found variations between studies in some polymorphism, for example. The TLR1 (rs 5743618), TLR2 (rs5743708), TLR4 Asp299Gly, TLR4 Thr399Ile, TLR4 (rs7873784), TLR6 (rs5743810), TLR9 (rs5743836) was associated with the protection against TB. Meta-analysis was performed between polymorphisms and pulmonary tuberculosis to define increase or decrease in susceptibility to tuberculosis in various populations, which indicated that a relationship exists between SNPs/host genetic factors and susceptibility or resistance in patients suffering from pulmonary tuberculosis our finding concludes that this gene polymorphism may be associated with susceptibility to TB. The present study adds value to the various researches and studies going on various populations of the world in better understanding the role of TLR polymorphism in TB.

Effects of prenatal opioid and alcohol exposures on immune and serotonin factors in human placenta

PurposeOpioids and alcohol impact critical serotonin (5-HT) function in the developing placenta and fetus through the actions of immune proinflammatory factors. Yet, possible convergent effects of opioids and alcohol on human placental toll-like receptor 4 (TLR4) activation and subsequent 5-HT homeostasis remain entirely unknown. The purpose of this study was to examine the effect of prenatal exposure to opioids with or without prenatal alcohol exposure (PAE) on the expression of key placental immune and serotonin signaling factors in human placental tissue obtained from a well-characterized prospective cohort. MethodsData were collected from a subset of participants enrolled in the prospective pre-birth Ethanol, Neurodevelopment, Infant, and Child Health (ENRICH-1) cohort. Women were recruited and classified into four study groups: 1) PAE (n = 20); 2) those taking medications for opioid use disorder (MOUD; n = 28), 3) concurrent PAE and MOUD (n = 20); and 4) controls (HC; n = 20) based on prospective, repeated self-report, and biomarker analysis. Placenta samples underwent tissue processing to identify mRNA for TLR4, nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), serotonin transporter (SERT), tryptophan hydroxylase (TPH1), indoleamine 2,3-Dioxygenase 1 (IDO) as well as protein concentrations of TLR4, IL-1β, TNF-α, SERT. To consider the association between study group and mRNA/protein expression of our targets, multivariable regression models were developed with inclusion of a priori selected covariates. ResultsThere was a significant negative association between PAE and SERT mRNA (β = −0.01; p < 0.01) and a positive association with TPH1 mRNA expression (β = 0.78; p < 0.05). In addition, there was a negative association between MOUD and TNF-α protein expression (β = −0.12; p < 0.05). ConclusionsThis study provides the first evidence that PAE may inhibit SERT expression while simultaneously promoting increased TPH1 protein expression in human placenta. This may result in increased 5-HT in fetal circulation known to affect neurodevelopment. Our data suggest that opioids and alcohol may disturb the bidirectional, dynamic interaction between the placental immune and serotonin system. Given the implication for brain development and health across the life-span further investigation of these critical mechanisms in well-defined cohorts is required.

Signal Sequence-Dependent Orientation of Signal Peptide Fragments to Exosomes.

Signal peptides (SPs) not only mediate targeting to the endoplasmic reticulum (ER) but also play important roles as biomarkers and substances with physiological activity in extracellular fluids including blood. SPs are thought to be degraded intracellularly, making it unclear how they are transported from the ER to the extracellular fluid. In a recent study, we showed that a C-terminal fragment of the SP of a type I membrane protein, amyloid precursor protein (APP), was secreted into the extracellular fluid via exosomes using transformed HEK293 cells expressing APP SP flanking a reporter protein. In the present study, we demonstrate that a N-terminal fragment of the SP from a type II membrane protein, human placental secreted alkaline phosphatase (SEAP), is contained in exosomes and secreted into the extracellular fluid using HEK-Blue hTLR3 cells, which express both a human toll-like receptor 3 gene and an inducible SEAP reporter gene. When HEK-Blue hTLR3 cells were stimulated with a TLR3 ligand, a N-terminal fragment of SEAP SP in exosomes was increased in parallel with SEAP secretion in a concentration-dependent manner. These results indicated that SP fragments are exosomal components. In addition, migrating SP fragments were determined by characteristics of the signal–anchor sequence of membrane proteins. Furthermore, we found that SP fragments could bind to calmodulin (CALM), which is a cytosolic protein and also a component of exosomes, suggesting its involvement in the transportation of SP fragments from the endoplasmic reticulum to exosomes.

Genome-Wide ChIP-seq and RNA-seq Analyses of STAT3 Target Genes in TLRs Activated Human Peripheral Blood B Cells.

Toll like receptors (TLRs) induced response plays a vital role in B-cell development and activation, in which TLR7-mediated and TLR9-mediated response interact together and play antagonistic or cooperative roles at different situations. Previous studies showed that the transcription factor signal transducer and activator of transcription (STAT) 3 was one of the key transcriptional factors (TFs) needed for both TLR7 and TLR9 signaling in B cell, and patients with autosomal dominant hyper IgE syndromes (AD-HIES) due to STAT3 mutations having defective TLRs response in B cells. However, how STAT3 affects its target genes and the downstream signaling pathways in B cell upon TLRs stimulation remains unclarified on a genome-wide level. ChIP-seq and RNA-seq was used in this study to identify the STAT3 targets in response to TLRs stimulation in human B cell. STAT3 ChIP-seq results showed a total of 611 and 2,289 differential STAT3-binding sites in human B cell after TLR7 and TLR9 agonists stimulation, respectively. RNA-seq results showed 1,186 and 1,775 differentially expressed genes after TLR7 and TLR9 activation, respectively. We identified 47 primary STAT3 target genes after TLR7 activation and 189 target genes after TLR9 activation in B cell by integration of STAT3 ChIP-seq and RNA-seq data. Among these STAT3 primary targets, we identified 7 TFs and 18 TFs for TLR7 and TLR9 response, respectively. Besides, we showed that STAT3 might regulate TLR9, but not TLR7 response in B cells through directly regulating integrin signaling pathway, which might further affect the antagonism between TLR7 and TLR9 signaling in B cell. Our study provides insights into the molecular mechanism of human TLRs response in B cell and how it can be regulated, which helps to better understand and modulate TLR-mediated pathogenic immune responses in B cell.

Fermentation of Plant Extracts Supplemented with Milk Components by Lactic Acid Bacteria Produces Soluble Agonists for Toll-like Receptor 2 Possibly Suitable for Cosmetics

Stimulation of Toll-like receptor 2 (TLR2) on epidermal keratinocytes results in the tightening of cell–cell junctions between keratinocytes; therefore, appropriate agonists for TLR2 could be promising ingredients for cosmetics. However, a method to produce significant amounts of soluble TLR2 agonists using materials that are suitable for preparing cosmetics has not yet been developed. In this study, we tried to identify appropriate lactic acid bacterial strains and media for fermentation to obtain soluble TLR2 agonists from traditional fermented foods and natural food sources. We found that Lactobacillus delbrueckii subsp. lactis TL24 (TL24) and a combination of hot water extracts of asparagus edible stem and cow skimmed milk were the best strain and culture medium, respectively, for this purpose. The TL24 ferments effectively stimulated TLR2 in HEK293 reporter cells expressing human TLR2 on their surface and also inhibited paracellular molecular transfer in a cell sheet of human primary keratinocytes. Since these effects of the TL24 ferments were suppressed by anti-TLR2 neutralizing antibodies, it is proposed that TL24 ferments elicit these effects via TLR2. Taken together, these results suggest that TL24 ferments containing soluble TLR2 agonists are potential ingredients for cosmetics.

In silico Designing of an Epitope-Based Vaccine Against Common E. coli Pathotypes.

Escherichia coli (E. coli) is a Gram-negative bacterium that belongs to the family Enterobacteriaceae. While E. coli can stay as an innocuous resident in the digestive tract, it can cause a group of symptoms ranging from diarrhea to live threatening complications. Due to the increased rate of antibiotic resistance worldwide, the development of an effective vaccine against E. coli pathotypes is a major health priority. In this study, a reverse vaccinology approach along with immunoinformatics has been applied for the detection of potential antigens to develop an effective vaccine. Based on our screening of 5,155 proteins, we identified lipopolysaccharide assembly protein (LptD) and outer membrane protein assembly factor (BamA) as vaccine candidates for the current study. The conservancy of these proteins in the main E. coli pathotypes was assessed through BLASTp to make sure that the designed vaccine will be protective against major E. coli pathotypes. The multitope vaccine was constructed using cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B cell lymphocyte (BCL) epitopes with suitable linkers and adjuvant. Following that, it was analyzed computationally where it was found to be antigenic, soluble, stable, and non-allergen. Additionally, the adopted docking study, as well as all-atom molecular dynamics simulation, illustrated the promising predicted affinity and free binding energy of this constructed vaccine against the human Toll-like receptor-4 (hTLR-4) dimeric state. In this regard, wet lab studies are required to prove the efficacy of the potential vaccine construct that demonstrated promising results through computational validation.

Pilus proteins from Streptococcus pyogenes stimulate innate immune responses through Toll-like receptor 2.

The group A Streptococcus (GAS) pilus is a long, flexible, hair‐like structure anchored to the cell surface that facilitates the adherence of GAS to host cells, thus playing a critical role in initiating infections. Because of its important role in GAS virulence, the pilus has become an attractive target for vaccine development. While current research mainly focuses on pilus function and its potential as a vaccine component, there is a lack of knowledge on how the host immune system recognizes and responds to this abundant surface structure. Here we show that both assembled GAS pili and individual pilus proteins induce a potent release of the proinflammatory cytokines tumor necrosis factor and interleukin‐8. We further show that the surface‐exposed backbone pilin and ancillary pilin 1 subunits are Toll‐like receptor 2 (TLR2) agonists. Using reporter cell lines coexpressing human TLR2 in combination with either TLR1 or TLR6, we determined that activation was mediated by the TLR2/TLR6 heterodimer. Finally, we used solid‐phase and flow cytometry binding assays to illustrate a direct interaction between the pilus subunits and TLR2. These results provide further support for the suitability of the pilus as a vaccine component and opens potential avenues for using GAS pili as an adjuvant or immune‐modulation agent.

Computational molecular characterization of p53 and Human TLRs interactions.

Toll-like receptors (TLRs) are one of the major sensors to regulate innate immunity. It is present in inactive form within immune cells. However, after recognizing the conserved region of the foreign body, it gets activated by the foreign body, such as bacteria, viruses, fungus, etc. Recently, it is reported that apart from participating in innate immunity, these TLRs also play an important role in apoptosis and cancer. Moreover, very few reported that it is cross-talk with p53 protein within the cell. P53 protein is a transcription factor for many cellular proteins involved in cellular transduction. It directly as well as indirectly regulates a wide variety of cellular processes such as apoptosis, senescence, cell cycle arrest, differentiation, and DNA repair and replication and cancer dynamics. Various studies reported genetic level interaction between p53 and TLRs. However, molecular interaction studies are still few reported. In the present work, we computationally characterized molecular interaction between p53 and toll-like receptors. We used open web resources for docking and analyzing the data. Our molecular docking and molecular dynamics simulation results suggest that there is a significant interaction between p53 and toll-like receptors. The study could important for the possible therapeutic intervention.

Molecular insights into the interaction between human nicotinamide phosphoribosyltransferase and Toll-like receptor 4

The secreted form of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes a key reaction in intracellular NAD biosynthesis, acts as a damage-associated molecular pattern triggering Toll-like receptor 4 (TLR4)-mediated inflammatory responses. However, the precise mechanism of interaction is unclear. Using an integrated approach combining bioinformatics and functional and structural analyses, we investigated the interaction between NAMPT and TLR4 at the molecular level. Starting from previous evidence that the bacterial ortholog of NAMPT cannot elicit the inflammatory response, despite a high degree of structural conservation, two positively charged areas unique to the human enzyme (the α1-α2 and β1-β2 loops) were identified as likely candidates for TLR4 binding. However, alanine substitution of the positively charged residues within these loops did not affect either the oligomeric state or the catalytic efficiency of the enzyme. The kinetics of the binding of wildtype and mutated NAMPT to biosensor-tethered TLR4 was analyzed. We found that mutations in the α1-α2 loop strongly decreased the association rate, increasing the KD value from 18 nM, as determined for the wildtype, to 1.3 μM. In addition, mutations in the β1-β2 loop or its deletion increased the dissociation rate, yielding KD values of 0.63 and 0.22 μM, respectively. Mutations also impaired the ability of NAMPT to trigger the NF-κB inflammatory signaling pathway in human cultured macrophages. Finally, the involvement of the two loops in receptor binding was supported by NAMPT-TLR4 docking simulations. This study paves the way for future development of compounds that selectively target eNAMPT/TLR4 signaling in inflammatory disorders.

Life-Threatening Enterovirus 71 Encephalitis in Unrelated Children with Autosomal Dominant TLR3 Deficiency.

Enterovirus A71 (EV71) causes a broad spectrum of childhood diseases, ranging from asymptomatic infection or self-limited hand-foot-and-mouth disease (HFMD) to life-threatening encephalitis. The molecular mechanisms underlying these different clinical presentations remain unknown. We hypothesized that EV71 encephalitis in children might reflect an intrinsic host single-gene defect of antiviral immunity. We searched for mutations in the toll-like receptor 3 (TLR3) gene. Such mutations have already been identified in children with herpes simplex virus encephalitis (HSE). We sequenced TLR3 and assessed the impact of the mutations identified. We tested dermal fibroblasts from a patient with EV71 encephalitis and aTLR3 mutation and other patients with known genetic defects of TLR3 or related genes, assessing the response of these cells to TLR3 agonist poly(I:C) stimulation and EV71 infection. Three children with EV71 encephalitis were heterozygous for rare mutations-TLR3 W769X, E211K, and R867Q-all of which were shown to affect TLR3 function. Furthermore, fibroblasts from the patient heterozygous for the W769X mutation displayed an impaired, but not abolished, response to poly(I:C). We found that TLR3-deficient and TLR3-heterozygous W769X fibroblasts were highly susceptible to EV71 infection. Autosomal dominant TLR3 deficiency may underlie severe EV71 infection with encephalitis. Human TLR3 immunity is essential to protect the central nervous system against HSV-1 and EV71. Children with severe EV71 infections, such as encephalitis in particular, should be tested for inborn errors of TLR3 immunity.

Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality.

Recent evidence suggests that several cattle breeds may be more resistant to infection with the zoonotic pathogen Mycobacterium bovis. Our data presented here suggests that the response to mycobacterial antigens varies in macrophages generated from Brown Swiss (BS) and Holstein Friesian (HF) cattle, two breeds belonging to the Bos taurus family. Whole genome sequencing of the Brown Swiss genome identified several potential candidate genes, in particular Toll-like Receptor-2 (TLR2), a pattern recognition receptor (PRR) that has previously been described to be involved in mycobacterial recognition. Further investigation revealed single nucleotide polymorphisms (SNP) in TLR2 that were identified between DNA isolated from cells of BS and HF cows. Interestingly, one specific SNP, H326Q, showed a different genotype frequency in two cattle subspecies, Bos (B.) taurus and Bos indicus. Cloning of the TLR2 gene and subsequent gene-reporter and chemokine assays revealed that this SNP, present in BS and Bos indicus breeds, resulted in a significantly higher response to mycobacterial antigens as well as tri-acylated lipopeptide ligands in general. Comparing wild-type and H326Q containing TLR2 responses, wild-type bovine TLR2 response showed clear, diminished mycobacterial antigen responses compared to human TLR2, however bovine TLR2 responses containing H326Q were found to be partially recovered compared to human TLR2. The creation of human:bovine TLR2 chimeras increased the response to mycobacterial antigens compared to the full-length bovine TLR2, but significantly reduced the response compared to the full-length human TLR2. Thus, our data, not only present evidence that TLR2 is a major PRR in the mammalian species-specific response to mycobacterial antigens, but furthermore, that there are clear differences between the response seen in different cattle breeds, which may contribute to their enhanced or reduced susceptibility to mycobacterial infection.

Subtractive genomic approach toward introduction of novel immunogenic targets against Clostridioides difficile: Thinking out of the box

Clostridioides difficile is one of the major causatives of nosocomial infections worldwide. Antibiotic-associated diarrhea, pseudomembranous colitis, and toxic megacolon are the most common forms of C. difficile infection (CDI). Considering the high antibiotic resistance of C. difficile isolates and the low efficacy of immunization with toxin-related vaccines, we suggested that surface-exposed and secreted proteins could be considered as potential immunogenic targets against CDI. Various immuninformatics databases were used to predict antigenicity, allergenicity, B-cell epitopes, MHC-II binding sites, conserved domains, prevalence and conservation of proteins among the most common sequence types, molecular docking, and immunosimulation of immunogenic targets. Finally, 16 proteins belonging to three functional groups were identified, including proteins involved in the cell wall and peptidoglycan layer (nine proteins), flagellar assembly (five proteins), spore germination (one protein), and a protein with unknown function. Molecular docking results showed that among all the mentioned proteins, WP_009892971.1 (Acd) and WP_009890599.1 (a C40 family peptidase) had the strongest interactions with human Toll-like receptor 2 (TLR-2) and TLR-4. This study proposes a combination of C. difficile toxoid (Tcd) and surface-exposed proteins such as Acd as a promising vaccine formulation for protection against circulating clinical strains of C. difficile.

Cholesterol-Rich Lipid Rafts as Platforms for SARS-CoV-2 Entry.

Since its appearance, the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), the causal agent of Coronavirus Disease 2019 (COVID-19), represents a global problem for human health that involves the host lipid homeostasis. Regarding, lipid rafts are functional membrane microdomains with highly and tightly packed lipid molecules. These regions enriched in sphingolipids and cholesterol recruit and concentrate several receptors and molecules involved in pathogen recognition and cellular signaling. Cholesterol-rich lipid rafts have multiple functions for viral replication; however, their role in SARS-CoV-2 infection remains unclear. In this review, we discussed the novel evidence on the cholesterol-rich lipid rafts as a platform for SARS-CoV-2 entry, where receptors such as the angiotensin-converting enzyme-2 (ACE-2), heparan sulfate proteoglycans (HSPGs), human Toll-like receptors (TLRs), transmembrane serine proteases (TMPRSS), CD-147 and HDL-scavenger receptor B type 1 (SR-B1) are recruited for their interaction with the viral spike protein. FDA-approved drugs such as statins, metformin, hydroxychloroquine, and cyclodextrins (methyl-β-cyclodextrin) can disrupt cholesterol-rich lipid rafts to regulate key molecules in the immune signaling pathways triggered by SARS-CoV-2 infection. Taken together, better knowledge on cholesterol-rich lipid rafts in the SARS-CoV-2-host interactions will provide valuable insights into pathogenesis and the identification of novel therapeutic targets.