Characterization of the host response to pichinde virus infection in the Syrian golden hamster by species-specific kinome analysis.

The Syrian golden hamster has been increasingly used to study viral hemorrhagic fever (VHF) pathogenesis and countermeasure efficacy. As VHFs are a global health concern, well-characterized animal models are essential for both the development of therapeutics and vaccines as well as for increasing our understanding of the molecular events that underlie viral pathogenesis. However, the paucity of reagents or platforms that are available for studying hamsters at a molecular level limits the ability to extract biological information from this important animal model. As such, there is a need to develop platforms/technologies for characterizing host responses of hamsters at a molecular level. To this end, we developed hamster-specific kinome peptide arrays to characterize the molecular host response of the Syrian golden hamster. After validating the functionality of the arrays using immune agonists of defined signaling mechanisms (lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α), we characterized the host response in a hamster model of VHF based on Pichinde virus (PICV(1)) infection by performing temporal kinome analysis of lung tissue. Our analysis revealed key roles for vascular endothelial growth factor (VEGF), interleukin (IL) responses, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and Toll-like receptor (TLR) signaling in the response to PICV infection. These findings were validated through phosphorylation-specific Western blot analysis. Overall, we have demonstrated that hamster-specific kinome arrays are a robust tool for characterizing the species-specific molecular host response in a VHF model. Further, our results provide key insights into the hamster host response to PICV infection and will inform future studies with high-consequence VHF pathogens.

Los niveles de expresión de los receptores toll-like 2 y 4 en neutrófilos se asocian con el pronóstico de los pacientes con ictus isquémico / The levels of expression of toll-like receptors 2 and 4 in neutrophils are associated with the prognosis of ischaemic stroke patients

Introducción. La isquemia cerebral desencadena una respuesta inflamatoria muy intensa que se caracteriza por la infiltración de leucocitos en el parénquima cerebral. Entre estas células las primeras en llegar al cerebro son los neutrófilos. A pesar de ello, el papel de los neutrófilos y de la expresión de sus receptores de inmunidad innata TLR2 y TLR4 (toll-like 2 y 4) en la isquemia cerebral no está claro. Objetivo. Estudiar la asociación entre los niveles de neutrófilos y la expresión de TLR2 y TLR4 en dichas células y el pronóstico de los pacientes con ictus isquémico. Pacientes y métodos. Incluimos a 110 pacientes con ictus isquémico de menos de 12 h de evolución. Como variables principales consideramos la escala de Rankin modificada a los tres meses (mal pronóstico > 2) y el volumen del infarto (medido mediante tomografía computarizada entre los días 4 y 7). Analizamos el porcentaje de neutrófilos y la expresión media de TLR2 y TLR4 en neutrófilos a través de citometría de flujo, al ingreso, a las 24 h, 72 h y a los 7 días. Resultados. La expresión de TLR4 en neutrófilos a las 72 h (odds ratio, OR = 2,1; intervalo de confianza del 95%, IC 95% = 1,4-3,2) y a los 7 días (OR = 3,2; IC 95% = 1,7-6,1) se asoció de forma independiente con el pronóstico de los pacientes y con el volumen del infarto a las 72 h (B = 5,4; IC 95% = 2,9-7,8) y a los 7 días (B = 7,0; IC 95% = 4,5-9,6). Conclusiones. Los neutrófilos a través de la expresión de su TLR4 se asocian con el pronóstico de los pacientes con ictus isquémico (AU)

Introduction. Cerebral ischaemia triggers a very intense inflammatory response that is characterised by the infiltration of leukocytes in the brain parenchyma. The first of these cells to reach the brain are the neutrophils. Nevertheless, the role played by neutrophils and the expression of their innate immunity receptors TLR2 and TLR 4 (toll-like receptors 2 and 4) in cerebral ischaemia is still not fully understood. Aim. To examine the association between the levels of neutrophils and the expression of TLR2 and TLR4 in those cells and the prognosis of ischaemic stroke patients. Patients and methods. The study involved a sample of 110 patients with an ischaemic stroke that had started less than 12 hours earlier. The main variables that were taken into account were the modified Rankin scale at three months (poor prognosis > 2) and the volume of the infarction (measured by means of computerised tomography between days 4 and 7). We analysed the percentage of neutrophils and the mean expression of TLR2 and TLR4 in neutrophils by means of flow cytometry on admission, at 24 h, at 72 h and at 7 days. Results. The TLR4 expression in neutrophils at 72 h (odds ratio, OR = 2.1; confidence interval of 95%, CI 95% = 1.4-3.2)and at 7 days (OR = 3.2; CI 95% = 1.7-6.1) was associated independently with the patients’ prognosis and with the volume of the infarction at 72 h (B = 5.4; CI 95% = 2.9-7.8) and at 7 days (B = 7.0; CI 95% = 4.5-9.6). Conclusions. Neutrophils are associated with the prognosis of patients with ischaemic stroke through the expression of their TLR4 (AU)

Chicken TLR21 is an innate CpG DNA receptor distinct from mammalian TLR9.

TLRs comprise a family of evolutionary conserved sensory receptors that respond to distinct classes of ligands. For one major evolutionary branch of TLRs, the ligands are still largely unknown. Here we report the cloning and function of one member of this group, chicken TLR21 (chTLR21). This TLR is absent in the human species but has homologs in fish and frog and displays similarity with mouse TLR13. Expression of chTLR21 in HEK293 cells resulted in activation of NF-kappaB in response to unmethylated CpG DNA, typically recognized by mammalian TLR9. Silencing of chTLR21 (but not chTLR4) in chicken macrophages inhibited the response to CpG-DNA (but not to LPS), indicating similar functionality of the endogenous receptor. ChTLR21 responded to human- and murine-specific TLR9 ligands, as well as to bacterial genomic DNA isolated from Salmonella enterica serovar Enteritidis. Confocal microscopy located chTLR21 in the same intracellular compartments as human TLR9. Inhibition of the chTLR21 response by the endosomal maturation inhibitor chloroquine suggested that the receptor is functional in endolysosomes, as known for TLR9. The analogous localization and function of the phylogenetically only distantly related chTLR21 and mammalian TLR9 suggest that during evolution different classes of TLRs have emerged that recognize the same type of ligands.

Expression, purification, and crystallization of Toll/interleukin-1 receptor (TIR) domains.

Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) play crucial roles in host innate immune response against microbial infections. These receptors share a conserved cytoplasmic domain, the Toll/interleukin-1 receptor (TIR) domain, which is required for signaling through these receptors. Structural information on the TIR domains will be essential for understanding the molecular basis for signal transduction by these receptors.

Predicting Toll-like receptor structures and characterizing ligand binding.

Toll-like receptor (TLR) ligand-binding domains comprise 18-25 tandem copies of a 24-residue motif known as the leucine-rich repeat (LRR). Unlike other LRR proteins, TLRs contain significant numbers of non-consensus LRR sequences, which makes their identification by computer domain search programs problematic. Here, we provide methods for identifying non-consensus LRRs. Using the location of these LRRs, hypothetical models are constructed based on the known molecular structures of homologous LRR proteins. However, when a hypothetical model for TLR3 is compared with the molecular structure solved by x-ray crystallography, the solenoid curvature, planarity, and conformations of the LRR insertions are incorrectly predicted. These differences illustrate how non-consensus LRR motifs influence TLR structure. Since the determination of molecular structures by crystallography requires substantial amounts of protein, we describe methods for producing milligram amounts of TLR3 extracellular domain (ECD) protein. The recombinant TLR3-ECD previously used to solve the molecular structure of TLR3-ECD has also been used to study the binding of TLR3-ECD to its ligand, double-stranded RNA (dsRNA). In the last section, we describe the preparation of defined TLR3 ligands and present methods for characterizing their interaction with TLR3-ECD.