Victoria Offord

Variation matters: TLR structure and species-specific pathogen recognition

Toll-like receptors (TLRs) are a family of pattern recognition receptors that are an important link between innate and adaptive immunity. Many vaccines incorporate ligands for TLRs as an adjuvant and are developed in rodent models, with the resulting data transferred to other species. Vaccine features can be improved markedly by emphasizing the biological relevance when evaluating other animal models for host-pathogen interaction and by taking greater advantage of the unique experimental opportunities that are offered by large animal, non-rodent models. Here, we aim to summarize our current knowledge of species-specific TLR responses and briefly discuss that vaccine efficacy in relevant host species might be improved by considering the species-specific TLR responses.

LRRfinder: a web application for the identification of leucine-rich repeats and an integrative Toll-like receptor database.

Homology modelling is considered the most accurate technique for computational prediction of protein structure. However, this technique comes with fundamental caveats of dependency on template quality, identification of structural features and accuracy of alignment. Leucine-rich repeats (LRRs) characterise a diverse family of proteins. Recently resolved structures reveal a highly conserved region in LRRs that assemble into the curved parallel beta-sheet lining the inner circumference of their solenoid structure. Thus, prediction of these structurally important regions is essential in the comparative modelling of LRR proteins and their interactions. Here, we describe the generation of tLRRdb, a database of selected Toll-like receptor (TLR) sequences with annotated co-ordinates. Derived from this is LRRfinder, a web application for the identification of LRRs within user-defined sequences to facilitate identification of structurally important regions, particularly relevant for protein-protein interaction studies and classification of novel sequences. LRRfinder is available at: www.lrrfinder.com.

Anti-apolipoprotein A-1 IgG in patients with myocardial infarction promotes inflammation through TLR2/CD14 complex.

Abstract.  Pagano S, Satta N, Werling D, Offord V, de Moerloose P, Charbonney E, Hochstrasser D, Roux‐Lombard P, Vuilleumier N (Geneva University Hospital and Faculty of Medicine, Geneva, Switzerland; Geneva University Hospital and Faculty of Medicine, Geneva, Switzerland; Royal Veterinary College, Hertfordshire, UK; St. Michael’s Hospital, Toronto, Canada; Geneva University Hospital and Faculty of Medicine, Geneva, Switzerland). Anti‐apolipoprotein A‐1 IgG in patients with myocardial infarction promotes inflammation through TLR2/CD14 complex. J Intern Med 2012; 272: 344–357.

Species‐specific PAMP recognition by TLR2 and evidence for species‐restricted interaction with Dectin‐1

TLRs mediate recognition of a wide range of microbial products, including LPS, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these, for example, C‐type lectins, is likely to cooperate with TLR signaling in defining inflammatory responses. In the present study, we examined the importance of the ECD and intracellular TIR domain of boTLR2 and huTLR2 to induce a species‐specific response by creating a chimeric TLR2 protein. Our results indicate that the strength of the response to any TLR2 ligand tested was dependent on the extracellular, solenoid structure, but not the intracellular TIR domain. Furthermore, we examined whether the recognition of two PAMPs by Dectin‐1, a CLR, depends on the interaction with TLR2 from the same species. TLR2 expression seemed to affect the Dectin‐1‐dependent production of CXCL8 to β‐glucan containing zymosan as well as Listeria monocytogenes. Furthermore, the interaction of Dectin‐1 with TLR2 seemed to require that both receptors are from the same species. Our data demonstrate that the differences in the TLR2 response seen between the bovine and human system depend on the ECD of TLR2 and that collaborative recognition of distinct microbial components by different classes of innate‐immune receptors is crucial in orchestrating inflammatory responses.

LRRfinder2.0: A webserver for the prediction of leucine-rich repeats

Leucine-rich repeats (LRRs) are versatile motifs present in more than 6000 proteins throughout the phylogenetic kingdom. Tandem LRRs generate a characteristic horseshoe with a diverse range of functions. Fulfilling a key role in the innate immune system, LRRs form the TLR and NOD-like receptor (NLR) pathogen-recognition domain. Host–pathogen interactions mediated by LRRs drive those involved in ligand recognition to become distinct from their consensus motif. Most LRRs range between 21 and 30 residues; however, large insertions in certain TLRs can generate repeats of over 60 amino acids. LRR variability makes them ideal for species-specific mediation of host-pathogen interactions. Teleost TLRs show large insertions, making cross-species alignments difficult without prior demarcation of their LRR motifs. We present LRRfinder2.0, a webserver for LRR prediction. LRRfinder2.0 utilizes scoring matrices comprising more than 60,000 LRR motifs from more than 200 species. The underlying TLR database tLRRdb contains more than 3500 manually annotated sequences, augmenting identification of irregular LRR motifs.

Global Gene Expression Profiling of Myeloid Immune Cell Subsets in Response to In Vitro Challenge with Porcine Circovirus 2b

Compelling evidence suggests that the early interaction between porcine circovirus 2 (PCV-2) and the innate immune system is the key event in the pathogenesis of Post-Weaning Multisystemic Wasting Syndrome (PMWS). Furthermore, PCV2 has been detected in bone-marrow samples, potentially enabling an easy spread and reservoir for the virus. To assess the gene-expression differences induced by an in-vitro PCV2b infection in different three different myeloid innate immune cell subsets generated from the same animal, we used the Agilent Porcine Gene Expression Microarray (V2). Alveolar macrophages (AMØs), monocyte-derived dendritic cells (MoDCs) and bone-marrow cells (BMCs) were generated from each animal, and challenged with a UK-isolate of a PCV2 genotype b-strain at a MOI of 0.5. Remarkably, analysis showed a highly distinct and cell-type dependent response to PCV2b challenge. Overall, MoDCs showed the most marked response to PCV2b challenge in vitro and revealed a key role for TNF in the interaction with PCV2b, whereas only few genes were affected in BMCs and AMØs. These observations were further supported by an enrichment of genes in the downstream NF-κB Signalling pathway as well as an up regulation of genes with pro-apoptotic functions post-challenge. PCV2b challenge increases the expression of a large number of immune-related and pro-apoptotic genes mainly in MoDC, which possibly explain the increased inflammation, granulomatous inflammation and lymphocyte depletion seen in PMWS-affected pigs.

Replacement of two aminoacids in the bovine Toll-like receptor 5 TIR domain with their human counterparts partially restores functional response to flagellin

Flagellin potently induces inflammatory responses in mammalian cells by activating Toll-like receptor (TLR) 5. Recently, we were able to show that stimulation of bovine TLR5 resulted in neither NFκB signalling nor CXCL8 production. Like other TLRs, TLR5 recruits signalling molecules to its intracellular TIR domain, leading to inflammatory responses. Analysis of available TLR5 sequences revealed substitutions in all artiodactyl sequences at amo acid (AA) position 798 and 799. Interestingly, a putative binding site for PI3K was identified at tyrosine 798 in the human TLR5 TIR domain, analogous to the PI3K recruitment domain in the IL-1 receptor. Mutation of the artiodactyl residues at position 798, 799 or both with their corresponding human counterparts partially restored the response of bovine (bo)TLR5 to flagellin as well as phosphorylation of PI3K. Together, our results suggest a potential lack of phosphorylation of F798 and H799 in boTLR5 partially explains the lack in observed response.

Two TIR-like domain containing proteins in a newly emerging zoonotic Staphylococcus aureus strain sequence type 398 are potential virulence factors by impacting on the host innate immune response

Staphylococcus aureus, sequence type (ST) 398, is an emerging pathogen and the leading cause of livestock-associated methicillin-resistant S. aureus infections in Europe and North America. This strain is characterized by high promiscuity in terms of host-species and also lacks several traditional S. aureus virulence factors. This does not, however, explain the apparent ease with which it crosses species-barriers. Recently, TIR-domain containing proteins (Tcps) which inhibit the innate immune response were identified in some Gram-negative bacteria. Here we report the presence of two proteins, S. aureus TIR-like Protein 1 (SaTlp1) and S. aureus TIR-like Protein 2 (SaTlp2), expressed by ST398 which contain domain of unknown function 1863 (DUF1863), similar to the Toll/IL-1 receptor (TIR) domain. In contrast to the Tcps in Gram-negative bacteria, our data suggest that SaTlp1 and SaTlp2 increase activation of the transcription factor NF-κB as well as downstream pro-inflammatory cytokines and immune effectors. To assess the role of both proteins as potential virulence factors knock-out mutants were created. These showed a slightly enhanced survival rate in a murine infectious model compared to the wild-type strain at one dose. Our data suggest that both proteins may act as factors contributing to the enhanced ability of ST398 to cross species-barriers.

Potential evidence for biotype-specific chemokine profile following BVDV infection of bovine macrophages.

Chemokines play a key role in initiating the innate and subsequently adaptive immune response by recruiting immune cells to the site of an infection. Monocytes/macrophages (MØ) are part of the first line of defence against invading pathogens, and have been shown to release a variety of chemokines in response to infection. Here, we reveal the early transcriptional response of MØ to infection with cytopathogenic (cp) and non-cytopathogenic (ncp) bovine viral diarrhoea strains (BVDV). We demonstrate up-regulation of several key chemokines of the CCL and CXCL families in MØ exposed to cpBVDV, but not ncpBVDV. In contrast, infection of MØ with ncpBVDV led to down-regulation of chemokine mRNA expression compared to uninfected cells. Data suggest that ncpBVDV can shut down production of several key chemokines that play crucial roles in the immune response to infection. This study helps to further our understanding of the pathogenesis of BVDV infection, highlighting biotype-specific cellular responses.

Structural characterisation of Toll-like receptor 1 (TLR1) and Toll-like receptor 6 (TLR6) in elephant and harbor seals.

Pinnipeds are a diverse clade of semi-aquatic mammals, which act as key indicators of ecosystem health. Their transition from land to marine environments provides a complex microbial milieu, making them vulnerable to both aquatic and terrestrial pathogens, thereby contributing to pinniped population decline. Indeed, viral pathogens such as influenza A virus and phocine distemper virus (PDV) have been identified as the cause of several of these mass mortality events. Furthermore, bacterial infection with mammalian Brucella sp. and methicillin-resistant Staphylococcus aureus strains have also been observed in marine mammals, posing further risk to both co-habiting endangered species and public health. During these disease outbreaks, mortality rates have varied amongst different pinniped species. Analyses of innate immune receptors at the host-pathogen interface have previously identified variants which may drive these species-specific responses. Through a combination of both sequence- and structure-based methods, this study characterises members of the Toll-like receptor (TLR) 1 superfamily from both harbour and elephant seals, identifying variations which will help us to understand these species-specific innate immune responses, potentially aiding the development of specific vaccine-adjuvants for these species.