Respiratory syncytial virus induces TLR3 protein and protein kinase R, leading to increased double-stranded RNA responsiveness in airway epithelial cells

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells, causing bronchiolitis, upper respiratory infections, asthma exacerbations, chronic obstructive pulmonary disease exacerbations, and pneumonia in immunocompromised hosts. A replication intermediate of RSV is dsRNA. This is an important ligand for both the innate immune receptor, TLR3, and protein kinase R (PKR). One known effect of RSV infection is the increased responsiveness of airway epithelial cells to subsequent bacterial ligands (i.e., LPS). In this study, we examined a possible role for RSV infection in increasing amounts and responsiveness of another TLR, TLR3. These studies demonstrate that RSV infection of A549 and human tracheobronchial epithelial cells increases the amounts of TLR3 and PKR in a time-dependent manner. This leads to increased NF-kappaB activity and production of the inflammatory cytokine IL-8 following a later exposure to dsRNA. Importantly, TLR3 was not detected on the cell surface at baseline but was detected on the cell surface after RSV infection. The data demonstrate that RSV, via an effect on TLR3 and PKR, sensitizes airway epithelial cells to subsequent dsRNA exposure. These findings are consistent with the hypothesis that RSV infection sensitizes the airway epithelium to subsequent viral and bacterial exposures by up-regulating TLRs and increasing their membrane localization.     

Inhibition of toll-like receptor 7- and 9-mediated alpha/beta interferon production in human plasmacytoid dendritic cells by respiratory syncytial virus and measles virus

Human plasmacytoid dendritic cells (PDC) are key sentinels alerting both innate and adaptive immune responses through production of huge amounts of alpha/beta interferon (IFN). IFN induction in PDC is triggered by outside-in signal transduction pathways through Toll-like receptor 7 (TLR7) and TLR9 as well as by recognition of cytosolic virus-specific patterns. TLR7 and TLR9 ligands include single-stranded RNA and CpG-rich DNA, respectively, as well as synthetic derivatives thereof which are being evaluated as therapeutic immune modulators promoting Th1 immune responses. Here, we identify the first viruses able to block IFN production by PDC. Both TLR-dependent and -independent IFN responses are abolished in human PDC infected with clinical isolates of respiratory syncytial virus (RSV), RSV strain A2, and measles virus Schwarz, in contrast to RSV strain Long, which we previously identified as a potent IFN inducer in human PDC (Hornung et al., J. Immunol. 173:5935-5943, 2004). Notably, IFN synthesis of PDC activated by the TLR7 and TLR9 agonists resiquimod (R848) and CpG oligodeoxynucleotide 2216 is switched off by subsequent infection by RSV A2 and measles virus. The capacity of RSV and measles virus of human PDC to shut down IFN production should contribute to the characteristic features of these viruses, such as Th2-biased immune pathology, immune suppression, and superinfection.     

Cutting edge: TLR2-mediated proinflammatory cytokine and chemokine production by microglial cells in response to herpes simplex virus

Recent studies indicate that TLRs are critical in generating innate immune responses during infection with HSV-1. In this study, we investigated the role of TLR2 signaling in regulating the production of neuroimmune mediators by examining cytokine and chemokine expression using primary microglial cells obtained from TLR2-/- as well as wild-type mice. Data presented here demonstrate that TLR2 signaling is required for the production of proinflammatory cytokines and chemokines: TNF-alpha, IL-1beta, IL-6, IL-12, CCL7, CCL8, CCL9, CXCL1, CXCL2, CXCL4, and CXCL5. CXCL9 and CXCL10 were also induced by HSV, but their production was not dependent upon TLR2 signaling. Because TLR2-/- mice display significantly reduced mortality and diminished neuroinflammation in response to brain infection with HSV, the TLR2-dependent cytokines identified here might function as key players influencing viral neuropathogenesis.     

Decreased Toll-like receptor 8 expression and lower TNF-alpha synthesis in infants with acute RSV infection

Background

Toll-like receptors (TLRs) are part of the innate immune system, able to recognize pathogen-associated molecular patterns and activate immune system upon pathogen challenge. Respiratory syncytial virus (RSV) is a RNA virus particularly detrimental in infancy. It could cause severe lower respiratory tract disease and recurrent infections related to inadequate development of anti-viral immunity. The reason could be inadequate multiple TLRs engagement, including TLR8 in recognition of single-stranded viral RNA and diminished synthesis of inflammatory mediators due to a lower expression.

Methods

Intracellular TLR8 expression in peripheral blood monocytes from RSV-infected infants was profiled and compared to healthy adults and age matched controls. Whether the observed difference in TLR8 expression is a transitory effect, infants in convalescent phase (4-6 weeks later) were retested. Specific TLR8-mediated TNF-α production in monocytes during an acute and convalescent phase was analyzed.

Results

RSV-infected and healthy infants had lower percentage of TLR8-expressing monocytes than healthy adults whereas decreased of TLR8 protein levels were detected only for RSV-infected infant group. Lower protein levels of TLR8 in monocytes from RSV-infected infants, compared to healthy infants, negatively correlated with respiratory frequency and resulted in lower TNF-α synthesis upon a specific TLR8 stimulation. In the convalescent phase, levels of TLR8 increased, accompanied by increased TNF-α synthesis compared to acute infection.

Conclusions

Lower TLR8 expression observed in monocytes, during an acute RSV infection, might have a dampening impact on early anti-viral cytokine production necessary to control RSV replication, and subsequently initiate an adaptive Th1 type immune response leading to severe disease in infected infants.     

Regulation of mammalian defensin expression by Toll-like receptor-dependent and independent signalling pathways

The immune system consists of innate and adaptive immune responses. The innate immune system confers non-specific protection against a large number of pathogens, hence, serving as the first line of defence. The innate immune system utilizes Toll-like receptors (TLRs) to recognize and bind pathogen-associated molecular patterns (PAMPs). Binding of PAMPs leads to TLR activation, which, in turn, initiates MAPK- or NF-kappaB-dependent cascades that culminate in a proinflammatory response. This response involves the secretion of cytokines, chemokines and broad-spectrum antibacterial substances, such as defensins. Increased defensin synthesis is also mediated by the activation of receptors other than TLRs, such as NOD2, IL-17R and PAR-2. This review summarizes the recently characterized signalling pathways leading to increased defensin synthesis as well as the pathway by which defensins activate TLRs on immature dendritic and memory T cells. Thus, not only do defensins eliminate pathogens, but they also recruit the adaptive immune system in instances of infection and/or inflammation.     

Innate immune recognition of respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the leading cause of respiratory infection in infants and young children. Severe clinical manifestation of RSV infection is a bronchiolitis, which is common in infants under six months of age. Recently, RSV has been recognized as an important cause of respiratory infection in older populations with cardiovascular morbidity or immunocompromised patients. However, neither a vaccine nor an effective antiviral therapy is currently available. Moreover, the interaction between the host immune system and the RSV pathogen during an infection is not well understood. The innate immune system recognizes RSV through multiple mechanisms. The first innate immune RSV detectors are the pattern recognition receptors (PRRs), including toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), and nucleotide-biding oligomerization domain (NOD)-like receptors (NLRs). The following is a review of studies associated with various PRRs that are responsible for RSV virion recognition and subsequent induction of the antiviral immune response during RSV infection. [BMB Reports 2014; 47(4): 184-191]     

Books

Book reviews in this article:
C ommunication and B ehavior of W hales . R. Payne (ed.).
S trandings : W ays T o S ave W hales . F. D. r obson .
O rders and F amilies of R ecent M ammals of T he W orld . S. Anderson and J. Knox Jones, Jr. (eds.).
S oviet -A merican C ooperative R esearch on M arine M ammals . V olume l—P innipeds . F. H. Fay and G. A. Fedoseev (eds.).
P athobiology of M arine M ammal D iseases . E. B. Howard (ed.).
S eals of T he W orld . (Second Edition.) J. E. Ring. British Museum and Cornell University Press.
T he G ray W hale : E schrichtius R obustus . M. L. Jones, S. L. Swartt and S. Leatherwood.
T he S ierra C lub H andbook of W hales and D olphins . S. Leatherwood and R. R. Reeves.     

The role of innate immune receptors in the control of Brucella abortus infection: toll-like receptors and beyond

Research into intracellular sensing of microbial products is an up and coming field in innate immunity. Toll-like receptors (TLRs) recognize Brucella spp. and bacterial components and initiate mononuclear phagocyte responses that influence both innate and adaptive immunity. Recent studies have revealed the intracellular signaling cascades involved in the TLR-initiated immune response to Brucella infection. TLR2, TLR4 and TLR9 have been implicated in host interactions with Brucella; however, TLR9 has the most prominent role. Further, the relationship between specific Brucella molecules and various signal transduction pathways needs to be better understood. MyD88-dependent and TRIF-independent signaling pathways are involved in Brucella activation of innate immune cells through TLRs. We have recently reported the critical role of MyD88 molecule in dendritic cell maturation and interleukin-12 production during B. abortus infection. This article discusses recent studies on TLR signaling and also highlights the contribution of NOD and type I IFN receptors during Brucella infection. The better understanding of the role by such innate immune receptors in bacterial infection is critical in host-pathogen interactions.     

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.     

Impact of curcumin on toll-like receptors

Toll-like receptors (TLRs) have a pivotal role in the activation of innate immune response and inflammation. TLRs can be divided into two subgroups including extracellular TLRs that recognize microbial membrane components (TLR1, 2, 4, 5, 6, and 10), and intracellular TLRs that recognize microbial nucleic acids (TLR3, 7, 8, and 9). Curcumin is a dietary polyphenol from Curcuma longa L. that is reputed to have diverse biological and pharmacological effects. Extensive research has defined the molecular mechanisms through which curcumin mediates its therapeutic effects. One newly defined and important target of curcumin is the TLR, where it exerts an inhibitory effect. In the current study, we focus upon the TLR antagonistic effect of curcumin and curcumin's therapeutic effect as mediated via TLR inhibition. The available evidence indicates that curcumin inhibits the extracellular TLR 2 and 4 and intracellular TLR9 and thereby exerts a therapeutic effect in diseases such as cancer, inflammation, infection, autoimmune, and ischemic disease. Curcumin effectively modulates the TLR response and thereby exerts its potent therapeutic effects.     

Early side effects of intrathoracic BCG therapy in patients with stage I squamous cell carcinoma,adenocarcinoma, or large cell lung cancer

Summary Data are presented on 179 stage I lung cancer patients subjected to resection operations and then given adjuvant intrapleural BCG and subsequent isoniazid (INH) therapy and on 167 control patients given intrapleural saline and placebo pills in a two-arm randomized double-blind study. The predominant immediate response to BCG/INH therapy was hyperpyrexia, which was found to be more pronounced in patients with larger induration in pretreatment PPD skin tests. Subsequently, chemical hepatitis (6 cases after BCG/INH versus 1 case after saline/placebo), peripheral neuropathy (3 versus 1), dermatitis/hives (5 versus 2), pleural thickening (4 versus 0), and persistent fever (10 versus 0) were noted. Analysis of laboratory changes measured at 18 weeks following randomization revealed that patients with BCG/INH lost 1.1 kg in weight and 0.30 g/dl in hemoglobin concentration on average, whereas control patients gained 1.2 kg and 0.33 g/dl, respectively. Modest rises in SGOT and alkaline phosphatase were apparent at 6 weeks after instillation of BCG compared with controls, but these differences were no longer statistically signifikant after 18 weeks. These side effects notwhithstanding, the BCG/INH therapy was well tolerated.Members of the Lung Cancer Study Group include R. T. Eagan*, R. E. Lee, W. S. Payne, R. E. Ritts, and L. Weiland from the Mayo Clinic, Rochester; C. F. Mountain*, H. T. Barkley, O. H. Frazier, K. Hermes, E. Hersh, and M. Valdivieso from M.D. Anderson Hospital, Houston; L. D. Hill*, M. D. Hafermann, and E. Morgan from The Mason Clinic, Seattle; P. W. Wright* and K.-E. Hellstrom from the Hutchinson Cancer Center, Seattle; C. Bagley, L. P. Johnson, H. Kellogg, and R. D. Pinkham from the Swedish Medical Center, Seattle; T. D. Ivey from University Hospital, Seattle; S. Hammar from Virginia Mason Hospital, Seattle; W. Nelems from St. Paul's Hospital, Vancouver; R. Feld*, D. Bergsagel, T. C. Brown, J. Curtis, C. Keen, J. F. Pringle, I. Quirt, and L. Yeoh from The Princess Margaret Hospital, Toronto; M. Blackstein and M. Goldberg from Mount Sinai Hospital, Toronto; F. G. Pearson*, D. W. Chamberlain, J. Cooper, W. Evans, and T. Todd from Toronto General Hospital, Toronto; M. Baker and R. Ginsberg from Toronto Western Hospital, Toronto; R. I. Mitchell from Wellesley Hospital, Toronto; E. C. Holmes*, W. F. Coulson, K. P. Ramming, and T. H. Weisenburger from the University of California, Los Angeles; Z. Petrovich from Wadsworth Veterans Hospital, Los Angeles; R. K. Oldham*, J. T. Forbes, F. A. Greco, D. L. Page, R. Prager, R. L. Richardson, and S. L. Stroup from Vanderbilt University, Nashville; J. M. Lukeman* and S. M. Sajjad from the Pathology Reference Center of M.D. Anderson Hospital, Houston; P. Grifone, A. Lebeck, and T. Voss from the Operations Office, Silver Spring, Maryland; M. Gail, W. McGuire, J. Allegra, and L. Rubinstein from the National Cancer Institute, Bethesda, Maryland; and L. Eirich, W. Heineman, and J. Beach from Information Management Services, Bethesda, Maryland. Asterisks designate principal investigators.     

Mycobacterium abscessus glycopeptidolipid prevents respiratory epithelial TLR2 signaling as measured by HβD2 gene expression and IL-8 release

Mycobacterium abscessus has emerged as an important cause of lung infection, particularly in patients with bronchiectasis. Innate immune responses must be highly effective at preventing infection with M. abscessus because it is a ubiquitous environmental saprophyte and normal hosts are not commonly infected. M. abscessus exists as either a glycopeptidolipid (GPL) expressing variant (smooth phenotype) in which GPL masks underlying bioactive cell wall lipids, or as a variant lacking GPL which is immunostimulatory and invasive in macrophage infection models. Respiratory epithelium has been increasingly recognized as playing an important role in the innate immune response to pulmonary pathogens. Respiratory epithelial cells express toll-like receptors (TLRs) which mediate the innate immune response to pulmonary pathogens. Both interleukin-8 (IL-8) and human β-defensin 2 (HβD2) are expressed by respiratory epithelial cells in response to toll-like receptor 2 (TLR2) receptor stimulation. In this study, we demonstrate that respiratory epithelial cells respond to M. abscessus variants lacking GPL with expression of IL-8 and HβD2. Furthermore, we demonstrate that this interaction is mediated through TLR2. Conversely, M. abscessus expressing GPL does not stimulate expression of IL-8 or HβD2 by respiratory epithelial cells which is consistent with "masking" of underlying bioactive cell wall lipids by GPL. Because GPL-expressing smooth variants are the predominant phenotype existing in the environment, this provides an explanation whereby initial M. abscessus colonization of abnormal lung airways escapes detection by the innate immune system.     

Differential recognition of TLR-dependent microbial ligands in human bronchial epithelial cells

Bronchial epithelial cells represent the first line of defense against invading airborne pathogens. They are important contributors to innate mucosal immunity and provide a variety of antimicrobial effectors. However, mucosal surfaces are prone to contact with pathogenic, as well as nonpathogenic microbes, and therefore, immune recognition principles have to be tightly controlled to avoid uncontrolled permanent activation. TLRs have been shown to recognize conserved microbial patterns and to mediate inducible activation of innate immunity. Our experiments demonstrate that bronchial epithelial cells express functional TLR1-6 and TLR9 and thus make use of a common principle of professional innate immune cells. Although it was observed that TLR2 ligands dependent on heterodimeric signaling either with TLR1 or TLR6 were functional, other ligands like lipoteichoic acid were not. Additionally, it was found that bronchial epithelial cells could be stimulated only marginally by Gram-positive bacteria bearing known TLR2 ligands while Gram-negative bacteria were easily recognized. This correlated with low expression of TLR2 and the missing expression of the coreceptor CD36. Transgenic expression of both receptors restored responsiveness to the complete set of TLR2 ligands and Staphylococcus aureus. Additional gene-array experiments confirmed hyporesponsiveness to this bacterium while Pseudomonas aeruginosa and respiratory syncytial virus induced common, as well as pathogen-specific, sets of genes. The findings indicate that bronchial epithelium regulates its sensitivity to recognize microbes by managing receptor expression levels. This could serve the special needs of controlled microbial recognition in mucosal compartments.     

TLR signaling tailors innate immune responses in human microglia and astrocytes

The specific signals mediating the activation of microglia and astrocytes as a prelude to, or consequence of, CNS inflammation continue to be defined. We investigated TLRs as novel receptors mediating innate immune responses in human glial cells. We find that microglia express mRNA for TLRs 1-9, whereas astrocytes express robust TLR3, low-level TLR 1, 4, 5, and 9, and rare-to-undetectable TLR 2, 6, 7, 8, and 10 mRNA (quantitative real-time PCR). We focused on TLRs 3 and 4, which can signal through both the MyD88-dependent and -independent pathways, and on the MyD88-restricted TLR2. By flow cytometry, we established that microglia strongly express cell surface TLR2; TLR3 is expressed at higher levels intracellularly. Astrocytes express both cell surface and intracellular TLR3. All three TLRs trigger microglial activation upon ligation. TLR3 signaling induces the strongest proinflammatory polarizing response, characterized by secretion of high levels of IL-12, TNF-alpha, IL-6, CXCL-10, and IL-10, and the expression of IFN-beta. CXCL-10 and IL-10 secretion following TLR4 ligation are comparable to that of TLR3; however, other responses were lower or absent. TLR2-mediated responses are dominated by IL-6 and IL-10 secretion. Astrocytes respond to TLR3 ligation, producing IL-6, CXCL-10, and IFN-beta, implicating these cells as contributors to proinflammatory responses. Initial TLR-mediated glial activation also regulates consequent TLR expression; while TLR2 and TLR3 are subject to positive feedback, TLR4 is down-regulated in microglia. Astrocytes up-regulate all three TLRs following TLR3 ligation. Our data indicate that activation of innate immune responses in the CNS is not homogeneous but rather tailored according to cell type and environmental signal.     

Toll-like receptor agonists synergize with CD40L to induce either proliferation or plasma cell differentiation of mouse B cells

In a classical dogma, pathogens are sensed (via recognition of Pathogen Associated Molecular Patterns (PAMPs)) by innate immune cells that in turn activate adaptive immune cells. However, recent data showed that TLRs (Toll Like Receptors), the most characterized class of Pattern Recognition Receptors, are also expressed by adaptive immune B cells. B cells play an important role in protective immunity essentially by differentiating into antibody-secreting cells (ASC). This differentiation requires at least two signals: the recognition of an antigen by the B cell specific receptor (BCR) and a T cell co-stimulatory signal provided mainly by CD154/CD40L acting on CD40. In order to better understand interactions of innate and adaptive B cell stimulatory signals, we evaluated the outcome of combinations of TLRs, BCR and/or CD40 stimulation. For this purpose, mouse spleen B cells were activated with synthetic TLR agonists, recombinant mouse CD40L and agonist anti-BCR antibodies. As expected, TLR agonists induced mouse B cell proliferation and activation or differentiation into ASC. Interestingly, addition of CD40 signal to TLR agonists stimulated either B cell proliferation and activation (TLR3, TLR4, and TLR9) or differentiation into ASC (TLR1/2, TLR2/6, TLR4 and TLR7). Addition of a BCR signal to CD40L and either TLR3 or TLR9 agonists did not induce differentiation into ASC, which could be interpreted as an entrance into the memory pathway. In conclusion, our results suggest that PAMPs synergize with signals from adaptive immunity to regulate B lymphocyte fate during humoral immune response.     

Respiratory Syncytial Virus Limits α Subunit of Eukaryotic Translation Initiation Factor 2 (eIF2α) Phosphorylation to Maintain Translation and Viral Replication

The impact of respiratory syncytial virus (RSV) on morbidity and mortality is significant in that it causes bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and pneumonia in immunocompromised hosts. RSV activates protein kinase R (PKR), a cellular kinase relevant to limiting viral replication (Groskreutz, D. J., Monick, M. M., Powers, L. S., Yarovinsky, T. O., Look, D. C., and Hunninghake, G. W. (2006) J. Immunol. 176, 1733–1740). It is activated by autophosphorylation, likely triggered by a double-stranded RNA intermediate during replication of the virus. In most instances, ph-PKR targets the α subunit of eukaryotic translation initiation factor 2 (eIF2α) protein via phosphorylation, leading to an inhibition of translation of cellular and viral protein. However, we found that although ph-PKR increases in RSV infection, significant eIF2α phosphorylation is not observed, and inhibition of protein translation does not occur. RSV infection attenuates eIF2α phosphorylation by favoring phosphatase rather than kinase activity. Although PKR is activated, RSV sequesters PKR away from eIF2α by binding of the kinase to the RSV N protein. This occurs in conjunction with an increase in the association of the phosphatase, PP2A, with eIF2α following PKR activation. The result is limited phosphorylation of eIF2α and continued translation of cellular and viral proteins.     

Toll-like receptor 2 and Toll-like receptor 4 expression in human adrenals.

Toll-like receptors (TLRs) are key elements in the innate immune response, functioning as pattern-recognition receptors for the detection and response to endotoxins and other microbial ligands. Inflammatory cytokines play an important role in the activation of the hypothalamic-pituitary-adrenal HPA axis during inflammation and sepsis. The newly recognized major role of TLR2 and TLR4 and the adrenal stress response during critical illnesses such as inflammation and sepsis demand comprehensive analysis of their interactions. Therefore, we analyzed TLR2 and TLR4 expression in human adrenal glands. Western blot analysis demonstrated the expression of TLR2 and TLR4 in the human adrenocortical cell line NCI-H295. Immunohistochemical analysis of normal human adrenal glands revealed TLR2 and TLR4 expression in the adrenal cortex, but not in the adrenal medulla. Considering the crucial role of the HPA axis and the innate immune response during acute sepsis or septic shock, elucidating the functional interaction of these systems should be of great clinical relevance.