Transmembrane Toll-like Receptors Research Articles

TLR4 expression in the saliva of systemic lupus erythematosus correlated with the serum vitamin D/25-hydroxyvitamin D: A cross-sectional study

Aim: This study aimed to examine the correlation between serum vitamin D (25-hydroxyvitamin D [25(OH)D]) and the transmembrane Toll-like receptor (TLR) 4 expression on CD11b+ cells in systemic lupus erythematosus (SLE) saliva. Materials and Methods: A cross-sectional study was conducted on 30 SLE patients who regularly visited to rheumatology clinic and met the following criteria: no systemic diseases, no smoking, no drinking and use of oral contraceptives, no steroid and immunosuppressant medications, no pregnancy, no wearing of dentures, and orthodontic appliances. The subject’s blood samples were taken for electrochemiluminescence immunoassay analyzer methods of examining 25(OH)D level. Full unstimulated whole saliva collection for the measurement of TLR4 expression. The descriptive demographic data, the chi-square, t test, and linear regression (95% confidence level) were used to analyze the association between blood 25(OH)D concentrations and immune cells expressing TLR4 in saliva CD11b+ cells. Results: SLE subjects had an average blood concentration of 25(OH)D of 9.9 ± 4.6 ng/mL. in saliva CD11b+ cells, TLR4 expression was 26.0 ± 20.9%. r = 0.473 and P < 0.05 indicated a correlation between the saliva CD11b+ cells expressing TLR4 and the serum level of 25(OH)D. Only 25(OH)D predicted TLR4 expression of the saliva CD11b+ cells. Conclusions: TLR4 expression of Saliva CD11b+ cells correlated with the concentrations of serum 25(OH)D in SLE. Hence, vitamin D/25(OH)D may control the immune response of SLE patients’ oral cavity.

A CD36 transmembrane domain peptide interrupts CD36 interactions with membrane partners on macrophages and inhibits atherogenic functions

CD36 is a transmembrane glycoprotein receptor for oxidized low density lipoprotein (LDL) and other endogenous danger signals and promotes athero-thrombotic processes. CD36 has been shown to associate physically with other transmembrane proteins, including integrins, tetraspanins, and toll-like receptors, which modulate CD36-mediated cell signaling. The CD36 N-terminal transmembrane domain (nTMD) contains a GXXXG sequence motif that mediates protein-protein interactions in many membrane proteins. We thus hypothesized that the nTMD is involved in CD36 interactions with other membrane proteins. CD36 interactions with partner cell surface proteins on murine peritoneal macrophages were detected with an immunofluorescence-based proximity ligation cross linking assay (PLA) and confirmed by immunoprecipitation/immunoblot. Prior to performing these assays, cells were incubated with a synthetic 29 amino acid peptide containing the 22 amino acid of CD36 nTMD or a control peptide in which the glycine residues in GXXXG motif were replaced by valines. In functional experiments, macrophages were preincubated with peptides and then treated with oxLDL to assess LDL uptake, foam cell formation, ROS formation and cell migration. CD36 nTMD peptide treated cells compared to untreated or control peptide treated cells showed decreased CD36 surface associations with tetraspanin CD9 and ameliorated pathologically important CD36 mediated responses to oxLDL, including uptake of DiI-labeled oxLDL, foam cell formation, ROS generation, and inhibition of migration.

Dietary dihydroartemisinin supplementation alleviates intestinal inflammatory injury through TLR4/NOD/NF-κB signaling pathway in weaned piglets with intrauterine growth retardation.

The aim of present study was to evaluate whether diets supplemented with dihydroartemisinin (DHA) could alleviate intestinal inflammatory injury in weaned piglets with intrauterine growth retardation (IUGR). Twelve normal birth weight (NBW) piglets and 12 piglets with IUGR were fed a basal diet (NBW-CON and IUCR-CON groups), and another 12 piglets with IUGR were fed the basal diet supplemented with DHA at 80 mg/kg (IUGR-DHA group) from 21 to 49 d of age. At 49 d of age, 8 piglets with similar body weight in each group were sacrificed. The jejunal and ileal samples were collected for further analysis. The results showed that IUGR impaired intestinal morphology, increased intestinal inflammatory response, raised enterocyte apoptosis and reduced enterocyte proliferation and activated transmembrane toll-like receptor 4 (TLR4)/nucleotide-binding and oligomerization domain (NOD)/nuclear factor-κB (NF-κB) signaling pathway. Dihydroartemisinin inclusion ameliorated intestinal morphology, indicated by increased villus height, villus height-to-crypt depth ratio, villus surface area and decreased villus width of piglets with IUGR (P < 0.05). Compared with NBW piglets, IUGR piglets supplemented with DHA exhibited higher apoptosis index and caspase-3 expression, and lower proliferation index and proliferating cell nuclear antigen expression in the intestine (P < 0.05). Dihydroartemisinin supplementation attenuated the intestinal inflammation of piglets with IUGR, indicated by increased concentrations of intestinal inflammatory cytokines and lipopolysaccharides (P < 0.05). In addition, DHA supplementation down-regulated the related mRNA expressions of TLR4/NOD/NF-κB signaling pathway and upregulated mRNA expressions of negative regulators of TLR4 and NOD signaling pathway in the intestine of piglets with IUGR (P < 0.05). Piglets in the IUGR-DHA group showed lower protein expressions of TLR4, phosphorylated NF-κB (pNF-κB) inhibitor α, nuclear pNF-κB, and higher protein expression of cytoplasmic pNF-κB in the intestine than those in the IUGR-CON group (P < 0.05). In conclusion, DHA supplementation could improve intestinal morphology, regulate enterocyte proliferation and apoptosis, and alleviate intestinal inflammation through TLR4/NOD/NF-κB signaling pathway in weaned piglets with IUGR.

Lateral diffusion of CD14 and TLR2 in macrophage plasma membrane assessed by raster image correlation spectroscopy and single\xa0particle tracking

The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; consequently, several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. The membrane-anchored receptor cluster of differentiation 14 (CD14) and the transmembrane toll-like receptor 2 (TLR2) are important receptors in the plasma membrane of macrophages that activate the intracellular signaling cascade in response to pathogenic stimuli. The aim of the present work was to compare the diffusion coefficients of CD14 and TLR2 on the apical and basal membranes of macrophages using two fluorescence-based methods: raster image correlation spectroscopy (RICS) and single particle tracking (SPT). In the basal membrane, the diffusion coefficients obtained from SPT and RICS were found to be comparable and revealed significantly faster diffusion of CD14 compared with TLR2. In addition, RICS showed that the diffusion of both receptors was significantly faster in the apical membrane than in the basal membrane, suggesting diffusion hindrance by the adhesion of the cells to the substrate. This finding highlights the importance of selecting the appropriate membrane (i.e., basal or apical) and corresponding method when measuring receptor diffusion in live cells. Accurately knowing the diffusion coefficient of two macrophage receptors involved in the response to pathogen insults will facilitate the study of changes that occur in signaling in these cells as a result of aging and disease.

Патофизиологические механизмы иммунологической деконтаминации вируса простого герпеса 1 типа из роговицы

В обзоре представлено описание патофизиологических механизмов герпесвирусной инфекции. Согласно данным медицинской статистики, вирусом простого герпеса 1 типа инфицировано большинство населения планеты. В развивающихся странах данный вирус является ведущей инфекционной причиной поражения роговицы. Также вирусу простого герпеса 1 типа отводится роль одного из факторов, приводящих к отторжению трансплантата роговицы. Вышеописанные патологические явления сопряжены с перестройкой клеточных систем в ответ на вирусное воздействие. Недавние открытия в данной области обнаружили значительный вклад трансмембранных и эндосомальных Toll-подобных рецепторов во врожденный противовирусный клеточный ответ. Показано, что эндосомальные Toll-подобные рецепторы 3 типа экспрессируются в кератоцитах только после их фенотипического перехода в фибробласты. Данная трансформация обычно происходит в результате механических и патогенных воздействий на роговицу. Изменение рецепторного состава клеток в ответ на герпесвирусную инвазию вызывает выработку интерферонов 1 типа - интерферона-альфа, интерферона-бета, и синтезу провоспалительных цитокинов, что приводит к вирусной деконтаминации. This review describes pathophysiological mechanisms of herpes virus infection in cornea cells. It has been previously reported that herpes simplex virus type 1 (HSV-1) infects most of the world’s population. In developing countries, HSV-1 is the leading infectious cause of corneal damage. Also, herpes simplex virus type 1 was assigned the role of one of the factors leading to rejection of the corneal transplant. These pathological phenomena are associated with restructuring of cellular systems in response to viral exposure. Recent discoveries have revealed a significant contribution of transmembrane and endosomal Toll-like receptors to the innate antiviral cell response. It is well known that endosomal Toll-like receptors-3 are expressed in keratocytes only after their phenotypic transformation to fibroblasts. This transformation usually occurs as a result of mechanical or infectious impact on the cornea. Changes in the receptor composition of cells as a response to herpes virus invasion is the main cause of type 1 interferons (interferon-alpha and interferon-beta) production and expression of proinflammatory cytokines, which leads to viral decontamination.

In silico screening of antigenic B-cell derived T-cell epitopes and designing of a multi-epitope peptide vaccine for Acinetobacter nosocomialis

Globally, antibiotic-resistant and tolerated bacterial isolates of Acinetobacter species are imposing high financial cost on health care systems and as such, molecular targets with promising immune protection could provide substantive benefits to human healthcare. Here, we performed an in silico based proteome-wide screening for antigenic B-cell derived T-cell epitopes and their following use to design a multi-epitope peptide vaccine that can effectively engage the host immune system against Acinetobacter nosocomialis SSA3 strain. Epitopes of the fimbrial biogenesis outer membrane usher FimD protein: YQQGINNYL and YRTNYTTVG were revealed appropriate for multi-epitope peptide construct designing. This protein has no homology to the host, essential to the pathogen survival and is localized at the pathogen surface. The predicted epitopes have high affinity for the highly expressed DRB*0101 allele in humans based on the lowest IC50 value in MHCPred and have an exo-membrane topology for efficient immune system recognition. The designed multi-epitope peptide vaccine is composed of the mentioned shortlisted antigenic epitopes linked to each other through a GPGPG linker, and an EAAAK linker that joined the multi-epitope peptide to the Cholera B subunit from Vibrio cholera as an adjuvant to increase vaccine construct antigenicity. The vaccine construct was docked and simulated with a transmembrane toll-like receptor (TLR4) that revealed construct stable binding with the TLR4 through the adjuvant, allowing the epitopes exposed to the host immune system essential for generating effective innate and long-lasting adaptive immunity. The designed multi-epitope peptide vaccine may prompt the development of a vaccine to control refractory and deleterious A. nosocomialis infections.

Contact allergen (PPD and DNCB)-induced keratinocyte sensitization is partly mediated through a low molecular weight hyaluronan (LMWHA)/TLR4/NF-κB signaling axis

Allergic contact dermatitis (ACD) is caused by topical exposure to chemical allergens. Keratinocytes play a key role in innate immunity, as well as in ACD progression. The transmembrane Toll-like receptor 4 (TLR4), strongly implicated in skin inflammation, has the ability to bind Damage Associated Molecular Patterns (DAMPs), like Low Molecular Weight Hyaluronan (LMWHA). Previously, we had determined that p-phenylenediamine (PPD) and 2,4-dinitrochlorobenzene (DNCB) modulate keratinocyte HA deposition in a manner correlated to their sensitization. In the present study, we aimed to investigate putative co-operation of HA and TLR4 in the process of PPD and DNCB-induced keratinocyte activation. Contact sensitizers were shown to significantly increase the expression of Hyaluronan Synthases (HAS) and TLR4 in NCTC2544 human keratinocytes, as demonstrated by western blot and Real-Time PCR. These data, in correlation to earlier shown enhanced HA degradation suggest that the contact sensitizers facilitate HA turnover of keratinocytes and increase the release of pro-inflammatory, LMWHA fragments. Treatment with exogenous LMWHA enhanced TLR4, HAS levels and Nuclear factor-kappa beta (NF-κΒ) activation. PPD, DNCB and LMWHA-effects were shown to be partly executed through TLR4 downstream signaling as shown by Real-Time, western blot, siRNA and confocal microscopy approaches. Specifically, PPD and DNCB stimulated the activation of the TLR4 downstream mediator NF-κB. Therefore, the shown upregulation of TLR4 expression is suggested to further facilitate the release of endogenous, bioactive HA fragments and sustain keratinocyte activation. In conclusion, keratinocyte contact allergen-dependent sensitization is partly mediated through a LMWHA/TLR4/ NF-κB signaling axis.

Nucleic acid-sensing TLRs and autoimmunity: novel insights from structural and cell biology.

Invasion of pathogenic microorganisms or tissue damage activates innate immune signaling receptors that sample subcellular locations for foreign molecular structures, altered host molecules, or signs of compartment breaches. Upon engagement of innate immune receptors an acute but transient inflammatory response is initiated, aimed at the clearance of pathogens and cellular debris. Among the molecules that are sensed are nucleic acids, which activate several members of the transmembrane Toll-like receptor (TLR) family. Inappropriate recognition of nucleic acids by TLRs can cause inflammatory pathologies and autoimmunity. Here, we review the mechanisms involved in triggering nucleic acid-sensing TLRs and indicate checkpoints that restrict their activation to endolysosomal compartments. These mechanisms are crucial to sample the content of endosomes for nucleic acids in the context of infection or tissue damage, yet prevent accidental activation by host nucleic acids under physiological conditions. Decoding the molecular mechanisms that regulate nucleic acid recognition by TLRs is central to understand pathologies linked to unrestricted nucleic acid sensing and to develop novel therapeutic strategies.

Establishing the Structural Rules for Ligand Recognition, Signaling and Assembly in Innate Immune Receptors

Type-I transmembrane Toll-like receptors (TLRs) are central to host defense against pathogens. Of the ten human TLRs, TLR4 is of particular interest given the complex mechanism required to recognize lipopolysaccharide (LPS) from the outer membranes of Gram-negative bacteria, culminating in transfer of LPS by membrane-bound CD14 to TLR4 in complex with the MD-2 coreceptor. Atomically detailed and enhanced sampling molecular simulations have recently enabled us to identify the allosteric switch in TLR4 activation; key to the switching mechanism is the remarkable conformational plasticity of the coreceptor, which we show is functionally conserved across an entire family of lipid recognition domains. As well as unravelling the experimentally-established structure-activity relationships of natural and synthetic LPS derivatives, we have combined simulations with biochemical data for TLR4 interaction with dietary fatty acids, challenging the long-accepted notion that the link between obesity, chronic inflammation, and insulin resistance occurs via this receptor. Related approaches have uncovered the mechanisms by which LPS derivatives are transferred from bacterial LPS aggregates to the CD14 coreceptor, and identified both the dynamic gating mechanism and final bound state of a hypothesized amino-terminal pocket, consistent with available NMR data. Finally, we have constructed multiscale models of truncated and full-length receptor libraries spanning the human TLR family, enabling observation of their transmembrane oligomerization. We correlate this with in vitro association and acceptor photobleaching FRET data for dimerization within live cells, helping to identify key stabilizing motifs and rationalize disruptive mutations. Our results reveal the structural basis for the tight coupling between extracellular, transmembrane, and intracellular domains required for higher order assembly, conformational change and adaptor recruitment. Collectively, these studies extend our mechanistic understanding of membrane receptor function, and pave the way for novel immunomodulatory therapeutic approaches.

Abstract 260: Interruption of Protein-Protein Interaction of CD36 With Other Proteins Alters CD36 Biological Functions

CD36 is a transmembrane glycoprotein belonging to the type 2 scavenger receptor family. On macrophages and platelets CD36 recognizes oxidized LDL and other endogenous danger signals and thereby transmits signals that promote athero-thrombotic processes. CD36 has been shown to associate physically with other transmembrane proteins, including integrins, tetraspanins, and toll-like receptors, and CD36-mediated cell signaling is modulated by these partner molecules. The CD36 N-terminal transmembrane domain (nTMD) contains a GXXXG sequence motif that in several membrane proteins has been shown to mediate protein-protein interactions. We thus hypothesized that the nTMD is involved in CD36 protein-protein interactions. We detected CD36 interactions with specific macrophage surface proteins with an immunofluorescence-based cross linking assay (Proximity Ligation Assay®/PLA) and confirmed them by immunoprecipitation/immunoblot (IP/IB). Prior to these assays cells were incubated with a synthetic 29 amino acid peptide containing the 22 amino acid of CD36 nTMD or a control peptide in which the glycine residues in GXXXG motif were replaced by valine. In functional experiments, macrophages were pre-incubated with peptides and then treated with oxLDL or native LDL prior to assessing LDL uptake at 1hr, foam cell formation after 24hr by oil red O staining, ROS generation by fluorescence probes, and cell migration in a modified Boyden Chamber Assay. CD36 nTMD peptide treated cells compared to untreated or control peptide treated cells had decreased CD36 surface associations with both CD9 and CD18 as detected by both PLA and IP/IB. Functional assays revealed that CD36 nTMD peptide significantly decreased DiI-labeled oxLDL uptake and decreased foam cell formation by 44% (p &lt;0.0001). This was associated with a 32% decrease in oxLDL-mediated ROS generation (p &lt; 0.001). OxLDL-induced inhibition of macrophage migration was inhibited by 84% (p &lt; 0.0001) by TMD treatment compared to the control peptide. In summary, these data support the hypothesis that CD36 nTMD mediates interaction of CD36 with other proteins such as CD9 and CD18, and that altering these interactions in macrophages ameliorates pathologically important CD36 mediated responses to oxLDL.

Combined Stimulation of Toll-Like Receptor 5 and NOD1 Strongly Potentiates Activity of NF-κB, Resulting in Enhanced Innate Immune Reactions and Resistance to Salmonella enterica Serovar Typhimurium Infection

Pathogen recognition receptors (PRRs) are essential components of host innate immune systems that detect specific conserved pathogen-associated molecular patterns (PAMPs) presented by microorganisms. Members of two families of PRRs, transmembrane Toll-like receptors (TLRs 1, 2, 4, 5, and 6) and cytosolic NOD receptors (NOD1 and NOD2), are stimulated upon recognition of various bacterial PAMPs. Such stimulation leads to induction of a number of immune defense reactions, mainly triggered via activation of the transcription factor NF-κB. While coordination of responses initiated via different PRRs sensing multiple PAMPS present during an infection makes clear biological sense for the host, such interactions have not been fully characterized. Here, we demonstrate that combined stimulation of NOD1 and TLR5 (as well as other NOD and TLR family members) strongly potentiates activity of NF-κB and induces enhanced levels of innate immune reactions (e.g., cytokine production) both in vitro and in vivo. Moreover, we show that an increased level of NF-κB activity plays a critical role in formation of downstream responses. In live mice, synergy between these receptors resulting in potentiation of NF-κB activity was organ specific, being most prominent in the gastrointestinal tract. Coordinated activity of NOD1 and TLR5 significantly increased protection of mice against enteroinvasive Salmonella infection. Obtained results suggest that cooperation of NOD and TLR receptors is important for effective responses to microbial infection in vivo.

MicroRNA Expression in Cystic Fibrosis Airway Epithelium.

MicroRNAs (miRs) have emerged as major regulators of the protein content of a cell. In the most part, miRs negatively regulate target mRNA expression, with sets of miRs predicted to regulate certain signaling pathways. The miR expression profile of endobronchial brushings is altered in people with cystic fibrosis (CF) compared to those without CF. How this impacts on CF has important implications for our growing understanding of the pathophysiology of CF lung disease and the development of new therapeutics to treat its pulmonary manifestations. Herein we discuss the potential consequences of altered miR expression in CF airway epithelium particularly with respect to cystic fibrosis transmembrane conductance regulator (CFTR) expression, innate immunity and toll-like receptor signalling and explore how best to exploit these changes for therapeutic benefit.

Loss of the ITAM receptor CD200R1 attenuates HSV-1 infection and encephalitis. (68.15)

Abstract We have discovered a novel role for the Immunoreceptor tyrosine-based activation motif (ITAM) containing receptor CD200R1 in virus infection. The host response to viral pathogens is driven by the activation of innate immune receptors that are triggered by pathogen-associated molecular pattern (PAMPs). These receptors include the transmembrane Toll-like receptors as well as the intracellular nucleic acid sensors RIG-I, Mda5, AIM2 and IFI16. ITAM receptors are known to both attenuate and activate signaling by innate receptors like TLRs but little is known about the potential impact of ITAMs on the outcome of viral infection. We generated CD200R1 knockout (KO) mice and infected with herpes simplex virus (HSV)-1. CD200R1 KO mice were protected from lethal HSV-1 encephalitis with &amp;gt;75% of the CD200R1 KOs surviving compared to 25% of the wild type (WT) mice, i.e., loss of CD200R1 conferred a survival advantage. This was not due to any obvious effects on the inflammatory response to infection. CD200R1 KO and WT mice had equivalent cytokine responses and leukocyte infiltration during the initial infection. Remarkably, HSV-1 virus replication was attenuated in CD200R1 KOs. Thus 50% of the CD200R1 KOs had cleared the virus from their brain by day 3-4 post-infection. In contrast, HSV-1 continued to replicate in the brains of 100% of the WT mice. Infection of cultured cells confirmed that CD200R1 expressing WT cells were more permissive for HSV-1 infection than CD200R1 KO cells.

Recognition of Streptococcus pneumoniae by the innate immune system

Streptococcus pneumoniae is both a frequent colonizer of the upper respiratory tract and a leading cause of life-threatening infections such as pneumonia, meningitis and sepsis. The innate immune system is critical for the control of colonization and for defence during invasive disease. Initially, pneumococci are recognized by different sensors of the innate immune system called pattern recognition receptors (PRRs), which control most subsequent host defence pathways. These PRRs include the transmembrane Toll-like receptors (TLRs) as well as the cytosolic NOD-like receptors (NLRs) and DNA sensors. Recognition of S. pneumoniae by members of these PRR families regulates the production of inflammatory mediators that orchestrate the following immune response of infected as well as neighbouring non-infected cells, stimulates the recruitment of immune cells such as neutrophils and macrophages, and shapes the adaptive immunity. This review summarizes the current knowledge of the function of different PRRs in S. pneumoniae infection.

The Molecular Basis for Lipid Recognition and Signaling in the Toll-Like Receptor Complex and a Family of Lipid-Binding Proteins

The transmembrane toll-like receptors (TLRs) are the initial gateway to almost all mammalian inflammatory responses to invading microbes. They are specialized for binding ligands with diverse structural and physiochemical properties ranging from microbial cell wall components to nucleic acids, and there is substantial interest in their pharmacological manipulation. TLR4 binds specifically to lipopolysaccharide (LPS), the central glycolipid component of Gram-negative bacterial outer membranes. Minute amounts of lipid A, the bioactive component of LPS, are an early sign of infection, and the association of TLR4 with many infectious and inflammatory diseases emphasizes its therapeutic importance. Subtle variations in the structure of lipid A profoundly affect activity, leading to unpredictable changes in TLR4 immune responses. To dissect the mechanisms of recognition and signaling, an extensive, explicitly-solvated, all-atom molecular dynamics simulation study has been performed for the isolated lipid-binding MD-2 co-receptor, and for the entire active, multimeric TLR4/MD-2 receptor complex, in the presence of a range of bound lipid A analogues, including several synthetic immunomodulatory mimetics undergoing clinical trials. We show that concerted large-scale conformational changes in MD-2 control the signaling status of the receptor complex, responding to variations in ligand acyl tail composition and headgroup phosphorylation status. Moreover, the co-receptor is part of a lipid-recognition superfamily, comprising immunoglobulin-like proteins involved in lipid signaling, transfer, and metabolism. Thus, we also report simulations of a wide range of these proteins, with or without ligands (including polyethers, fatty acids, single-chain lipids, and sterols), which reveal ligand-responsive conformational dynamics comparable to TLR4/MD-2. Therefore, a shared mechanism is identified in these distantly related family members, explaining variations in available structures obtained using different experimental approaches, and suggesting a molecular basis for allergic mimicry in dust-mite allergen lipid-binding proteins.

Essential role of high-mobility group box proteins in nucleic acid-mediated innate immune responses

Central to protective and pathological immunity is the activation of innate immune responses upon recognition of nucleic acids by transmembrane Toll-like receptors (TLRs) and cytosolic receptors. In mammals, the transmembrane pattern recognition receptors TLR3, TLR7 and TLR9 recognize double-stranded RNA, single-stranded RNA and hypomethylated DNA, respectively, while the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), RIG-I and MDA5 are known to be cytosolic RNA-sensing receptors. In addition, cytosolic DNA-sensing receptors that include DAI, RIG-I/MDA5 and AIM2 also trigger innate immune responses. High-mobility group box (HMGB)1, 2 and 3 proteins, which also bind immunogenic nucleic acids, are generally involved in the nucleic acid receptor-mediated activation of innate immune responses. There is a hierarchy in the nucleic acid-mediated activation of immune responses, wherein the selective activation of the nucleic acid-sensing receptors is contingent on the more promiscuous sensing of nucleic acids by HMGBs. The aim of this review is to summarize this novel feature of HMGB proteins, as essential frontline instigators of nucleic acid-mediated activation of innate immune responses. In addition, we will discuss the therapeutic implications of these findings.

Toll-like receptors in secondary obstructive cholangiopathy.

Secondary obstructive cholangiopathy is characterized by intra- or extrahepatic bile tract obstruction. Liver inflammation and structural alterations develop due to progressive bile stagnation. Most frequent etiologies are biliary atresia in children, and hepatolithiasis, postcholecystectomy bile duct injury, and biliary primary cirrhosis in adults, which causes chronic biliary cholangitis. Bile ectasia predisposes to multiple pathogens: viral infections in biliary atresia; Gram-positive and/or Gram-negative bacteria cholangitis found in hepatolithiasis and postcholecystectomy bile duct injury. Transmembrane toll-like receptors (TLRs) are activated by virus, bacteria, fungi, and parasite stimuli. Even though TLR-2 and TLR-4 are the most studied receptors related to liver infectious diseases, other TLRs play an important role in response to microorganism damage. Acquired immune response is not vertically transmitted and reflects the infectious diseases history of individuals; in contrast, innate immunity is based on antigen recognition by specific receptors designated as pattern recognition receptors and is transmitted vertically through the germ cells. Understanding the mechanisms for bile duct inflammation is essential for the future development of therapeutic alternatives in order to avoid immune-mediated destruction on secondary obstructive cholangiopathy. The role of TLRs in biliary atresia, hepatolithiasis, bile duct injury, and primary biliary cirrhosis is described in this paper.

Pattern recognition receptors and genetic risk for rsv infection: value for clinical decision‐making?

Respiratory syncytial virus (RSV) causes respiratory tract infections, especially among young infants. Practically, all infants are infected during epidemics and the clinical presentation ranges from subclinical to fatal infection. Known risk factors for severe RSV infection include prematurity, age of <2 months, underlying chronic lung or heart diseases, serious neurological or metabolic disorders, immune deficiency (especially a disorder of cellular immunity), crowded living conditions, and indoor smoke pollution. Twin studies indicate that host genetic factors affect susceptibility to severe RSV infection. Pattern recognition receptors (PRRs) are the key mediators of the innate immune response to RSV. In the distal respiratory tract, RSV is recognized by the transmembrane Toll-like receptor 4 (TLR4) and adapter proteins, which lead to production of proinflammatory cytokines and subsequent activation of the adaptive immune response. Surfactant proteins A and D are able to bind both RSV and TLR4, modulating the inflammatory response. Genetic variations in TLR4, SP-A, and SP-D have been associated with the risk of severe RSV bronchiolitis, but the results have varied between studies. Both the homozygous hyporesponsive 299Gly genotype of TLR4 and the non-synonymous SP-A and SP-D polymorphism influence the presentation of RSV infection. The reported relative risks associated with these markers are not robust enough to justify clinical use. However, current evidence indicates that innate immune responses including pattern recognition receptors (PRRs) and other components in the distal airways and airspaces profoundly influence the innate immune responses, playing a key role in host resistance to RSV in young infants. This information is useful in guiding efforts to develop better means to identify the high-risk infants and to treat this potentially fatal infection effectively.

Campylobacter jejuni activates NF-κB independently of TLR2, TLR4, Nod1 and Nod2 receptors

Campylobacter jejuni activates the host transcription factor NF-κB that regulates the expression of a number of genes involved in the inflammatory response to bacterial infection. Signaling pathways leading to NF-κB by pathogens and/or their products include transmembrane Toll-like receptors (TLRs) and intracellular receptors nucleotide-binding oligomerization domain proteins (Nods). This study was carried out to investigate the role of TLRs (TLR2 and TLR4) and Nods (Nod1 and Nod2) receptors in mediating NF-κB activation by C. jejuni. By means of transfecting receptors/molecules under study and measuring reporter gene activity, NF-κB activation and subsequent cytokine production by live, heat-killed C. jejuni, or boiled cell extract (BCE) were observed in a range of tissue culture cell lines. This activation is reduced upon transfection of cells with the dominant negative versions (DNV) of TLR-adaptor molecule MyD88. NF-κB activation was observed to be augmented in cell lines transfected with TLR2, Nod1, and Nod2 but not with TLR4. Additionally, NF-κB activation by C. jejuni was observed to be independent of Nod1 and Nod2 in cells transfected with DNV of these receptors. NF-κB activation pathway by C. jejuni may represent a novel mechanism utilising unknown receptors up-regulated by yet to be characterized active component(s). To our knowledge, such observations have not been previously reported for C. jejuni or any other food-borne pathogen.

Salvage effect of E5564, Toll‐like receptor 4 antagonist on d‐galactosamine and lipopolysaccharide‐induced acute liver failure in rats

The transmembrane protein Toll-like receptor 4 (TLR4), which exists mainly in macrophages such as Kupffer cells of the liver, plays an important role in recognizing and mediating macrophage activation and pro-inflammatory cytokine release. Activation of the pro-inflammatory cytokine cascade, including tumor necrosis factor-alpha (TNF-alpha), has a pivotal role in the progression of severe liver injury. D-galactosamine (GalN) and lipopolysaccharide (LPS)-induced liver injury in rats is an experimental model of fulminant hepatic failure, where TNF-alpha plays a central role in the progression of liver injury. E5564, a synthetic analogue of the lipid A component of endotoxin, inhibits endotoxin-stimulated inflammation and is under study for patients with sepsis. In the present study, we sought to explore the salvage effect of TLR4 antagonist E5564 on GalN+LPS-induced acute liver failure (ALF) in rats. ALF was induced in male Wistar rats by the intraperitoneal injection of GalN (500 mg/kg) and LPS (50 microg/kg). Immediately after GalN+LPS injection, rats were treated with intravenous injection of E5564 (3 mg/kg). The cumulative survival rates of GalN+LPS-induced ALF rats were compared between those with and without E5564 treatment. The intravenous injection of E5564 reduced the elevation of serum total bilirubin, aspartate aminotransferase, alanine aminotransferase and TNF-alpha levels in rats at 3 h after GalN+LPS injection, and improved the survival rate of GalN+LPS-induced ALF rats at 24 h (8% vs 43%). TLR4 antagonist E5564 reduced GalN+LPS-induced acute liver injury in rats and improved the overall survival rate of GalN+LPS-induced ALF rats. It may contribute to the treatment of ALF through blocking endotoxin-induced TNF-alpha overproduction of macrophages.