Abstract
TLRs (Toll-like receptors), NLRs (Nod-like proteins) and RLRs (RIG-like receptors), pathogens sensors of innate immunity conference organized by EuroSciCon hosted a large group of scientists from across Europe. The Chairs hoped the meeting would “provide an overview of these three families of receptors and provide the most recent advances in the area of innate immune pattern recognition”, with a view to informing future directions in this field. Eleven talks and six posters were presented; the Chairs gave a detailed introduction, followed by a detailed summation at the end. This article aims to describe the key topics highlighted.
Structure & function of pattern recognition receptors
Immunotherapeutic modulation of pattern recognition receptors (PRRs) in the treatment or prophylaxis of human disease relies upon a sound understanding of the structural interactions among cognate proteins involved in either recognizing or transducing the signal. The conference was reminded that since the late 1980s, over 10,000 papers have been published concerning the subject of pattern recognition in innate immunity (Nick Gay, University of Cambridge, UK). Dirk Werling (Royal Veterinary College, London, UK) discussed results suggesting high conservation between bovine and human Toll-like receptor (TLR)2 Toll/IL-1 receptor (TIR) domains, and hypothesized that cross-species differences in pathogen recognition, possibly resulting from differential glycosylation or hydrophobicity within leucine-rich repeats, may be determined extracellularly. Werling also discussed his group’s current focus on bovine TLR activity in human embryonic kidney cell lines.
Gay demonstrated closed complex death domain association of myeloid differentiation factor (MyD)88 and IL-1 receptor-associated kinase (IRAK)4, which his group hypothesized may be necessary for TLR4 signal transduction. Small-angle X-ray scattering and electron microscopy revealed this closed complex to be structurally similar to the death domain associations that form the PIDDosome (Gay). Their findings are supportive of the hypothesis that higher order oligomeric structure formation is driven by postreceptor TLR signaling.
Gay also discussed the hypothesis that TLR activation relies upon the association of C’terminal juxtamembrane domain associations of TLR dimers, which may be induced by charge characteristics in leucine-rich repeat regions and ligands. Using the example of immunomodulators used in human papillomavirus, basal cell carcinoma and other intraepithelial dysplasias, Gay discussed the charge hypothesis in the context of TLR8 activation. The conference was reminded that TLR8 is negatively charged, and as imiquimod becomes positively charged in a low-pH environment, this may be conducive to their interaction.
Sensing of bacteria
The realization that TLR sensing of bacterial components in humans contributed to the onset of many clinical features of sepsis reinforced the interest of immunologists in PRRs. Whilst PRR activation in viruses often appears to favor the host over the pathogen, bacterial sensing represents a reverse clinical paradigm. Rohini Rana (Imperial College, London, UK) discussed the structure and functioning of the TIR-like proteins from Yersinia pestis as demonstrated through a combination of pull-down assays and nuclear MRI techniques. Rana showed that the TIR-like proteins exhibit 20–30% conservation across bacteria, yet only 10% in humans. Rana additionally showed via pull-down assays that the TIR-like proteins from Y. pestis are able to interact with human MyD88, which often acts as a transducer after prior upstream TLR activation in humans.
The potential targets for immunomodulation in Francisella tularensis are yet to be determined. The conference heard how Richard Saint (Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire UK) used western blotting and quantified densitometry analysis to determine intracellular activity downstream of TLR binding over the natural history of F. tularensis infection in the murine model. Saint showed biphasic p38 and IRAK responses over 72 h. Many different explanations were proposed for the differential patterns of activation of these second messengers and further research is required.
Clare Bryant (University of Cambridge, UK) discussed the PRR-mediated response to a variety of Gram-negative and Gram-positive bacteria species across a range of animal models. Bryant reminded the conference that the response to lipopolysaccharide (LPS) and other bacterial pathogen-associated molecular patterns (PAMPs) is not conserved in terms of severity across host species; the level of communication between TLR4 and MD2 during the response to LPS may differ, with LPS often acting as an antagonist in certain species but as an agonist in others.
While many murine studies involve the use of death/survival curves, Bryant’s approach has favored sublethal intravenous doses of Salmonella typhimurium, which has allowed a longer period of observation and measurement with a view to evaluating potential salmonella-associated PAMPs for murine TLRs. The main S. typhi PAMPs currently under consideration by Bryant include LPS, unmethylated DNA, lipoproteins and flagellin, which murine TLR homo- or heterodimers may be detecting. Bryant showed that at sublethal doses of S. typhi, TLR4 appears to be most active in transducing the inflammatory response, yet increasing the dose to a lethal level recruits TLR2. Bryant’s findings support the concepts of activation thresholds in PRR activity and that a TLR-promoted antigen specific response is not critical for murine survival in salmonella infection.
Sensing of fungi
The importance of virulence factors versus modulated host cytokine secretion in fungal pathogenesis and the clinical picture of fungal infection are being determined. Comparing fungal pathogens globally, Candida albicans is most destructive in humans, which consequently made this species the focus of the fungi section of the conference. Neil Gow (University of Aberdeen, UK), discussed recent discoveries relating to extracellular interactions between fungus and host, as modeled using C. albicans, revealing that TLR4 has a higher specificity for C. albicans motifs compared with the previously elucidated family of lectin receptors known to be involved in the innate response to fungal pathogens.
The research focus of David Moyes (Kings College London, London, UK) is concerned with intracellular transduction of C. albicansdetection. Moyes targeted the TR146 human oral epithelial cell line, determining that the stronger arm of the biphasic intracellular signaling response (c-Fos) is interactive with C. albicans, and that MAPK phosphatase 1 activation may be more dependent on C. albicans hyphal patterns.
Olaf Gross (University of Laussane, Switzerland) discussed the important role for the receptor-associated kinase, Syk, in the transduction of fungal signals from immunoreceptor tyrosinebased activation motif (ITAM)-coupled PRRs such as NOD-like protein 3 (Nlrp3). Gross also showed that nlrp3 knockout mice were unable to mount an inflammasome-mediated response to C. albicans, before highlighting that PAMPs for many Nod-like receptor genes had not been fully characterized.
Sensing of viruses
Viral nucleic acid sequences, as natural ligands for RNA helicase activity, have highlighted a new area of therapeutic research. Steve Goodburn (University of London, UK) explored the notion of influenza A virus having a strategy to obscure its 5´ triphosphate group ribonucleic acid sequences from detection by RNA helicase (and potentially other PRRs), and considered the well-known fact that viruses need to counter the interferon response to succeed. Goodburn hypothesized that paramyxovirus V protein (PIV5) is somehow able to block interferon induction by blinding internal PRRs to dsRNA; melanoma-associated differentiation gene 5 (mda5) is clearly crucial in this response as cells are unable to induce interferon responses to PIV5 if mda5 is knocked out. Goodburn is currently investigating a role for LGP2, another RNA helicase, in augmenting the MDA5 response, as it is unknown whether LGP2 antagonism is critical factor for viral survival during replication.
Sensing in allergy
Lucia Fili (Laboratory of Immunology, Firenze, Italy) demonstrated how substituted adenine is able to act as a TLR7 ligand in order to induce a Th1- rather than a Th2-mediated immune response. Fili demonstrated that the conjugation of substituted adenine to components of Dermatophagoides pteronyssinus is able to downregulate a Th2-mediated inflammatory response, and that TLR7 modulation in this manner can switch allergy-biased Th2 T cells into Th1 or Th0 interferon-expressing cell types, as confirmed via NFkB-luciferase reporter plasmid studies. These findings have promising implications for possible immunotherapeutic management of dust-mite allergy.
Conclusion
The discoveries marking current hypotheses concerning the activity of the trinity of PRRs have led to several major developments in immunotherapy in the clinical setting, as noted by Gay and Kathy Triantafilou (University of Sussex, Brighton, UK) during the introduction by the Chairs. The overwhelming consensus at the conference was that a lack of viable anti-PRR antibodies in various animal models presents an obstacle in moving forward with research in innate immunity. The Chairs’ summation proposed that further characterization of PRR structure and protein interactions will create a clearer picture of potential targets in immunotherapeutics and vaccine development.
Acknowledgements
This meeting was organized by Euroscicon: www.euroscicon.com.
Financial & competing interests disclosure
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
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