Novel Immunomodulators from Hard Ticks Selectively Reprogramme Human Dendritic Cell Responses

Abstract

Hard ticks subvert the immune responses of their vertebrate hosts in order to feed for much longer periods than other blood-feeding ectoparasites; this may be one reason why they transmit perhaps the greatest diversity of pathogens of any arthropod vector. Tick-induced immunomodulation is mediated by salivary components, some of which neutralise elements of innate immunity or inhibit the development of adaptive immunity. As dendritic cells (DC) trigger and help to regulate adaptive immunity, they are an ideal target for immunomodulation. However, previously described immunoactive components of tick saliva are either highly promiscuous in their cellular and molecular targets or have limited effects on DC. Here we address the question of whether the largest and globally most important group of ticks (the ixodid metastriates) produce salivary molecules that specifically modulate DC activity. We used chromatography to isolate a salivary gland protein (Japanin) from Rhipicephalus appendiculatus ticks. Japanin was cloned, and recombinant protein was produced in a baculoviral expression system. We found that Japanin specifically reprogrammes DC responses to a wide variety of stimuli in vitro, radically altering their expression of co-stimulatory and co-inhibitory transmembrane molecules (measured by flow cytometry) and their secretion of pro-inflammatory, anti-inflammatory and T cell polarising cytokines (assessed by Luminex multiplex assays); it also inhibits the differentiation of DC from monocytes. Sequence alignments and enzymatic deglycosylation revealed Japanin to be a 17.7 kDa, N-glycosylated lipocalin. Using molecular cloning and database searches, we have identified a group of homologous proteins in R. appendiculatus and related species, three of which we have expressed and shown to possess DC-modulatory activity. All data were obtained using DC generated from at least four human blood donors, with rigorous statistical analysis. Our results suggest a previously unknown mechanism for parasite-induced subversion of adaptive immunity, one which may also facilitate pathogen transmission.

De novo assembly and annotation of the salivary gland transcriptome of Rhipicephalus appendiculatus male and female ticks during blood feeding

“…Similar to previous studies -which found male-specific expression for HBPM (Paesen et al, 1999) and IGBP-M (Wang and Nuttall, 1995) genes and female-specific expression for HBP1, HBP2 (Paesen et al, 1999), Kunitz/BPTI-like protein, Ra-KLP (Paesen et al, 2009) and Japanin (Preston et al, 2013) genes -we found malespecific expression for HBPM and IGBP-Ms and female-specific expression for HBP1, HBP2, Ra-KLP and Japanin in our transcriptome. Expression profiling corroborated the publically available knowledge of R. appendiculatus genes.…”

“…This was similar to what was shown for Japanin (Preston et al, 2013). The conserved cysteine residues and the tick Lipocalin motive, previously characterised in Japanin, were also observed in Rapp_Mc9023.…”

“…Protein identities between 48% and 88% were observed for four proteins; Serine proteinase inhibitors RAS-1 and -4 (Mulenga et al, 2003b), Male-specific histamine-binding protein, HBPM (Paesen et al, 1999) and Japanin (Preston et al, 2013). These four proteins represented 16% of the previously characterised proteins, which was in range with the percent identity observed between the R. appendiculatus transcriptome and the RaGI gene set (15% of best BLAST hits had identities ≤ 90%).…”

“…Additionally, the protein classes in each transcriptome were compared. A further comparison of the proteins assembled in this study against proteins previously identified as quantitative reverse transcriptase (RT) PCR reference genes (Nijhof et al, 2009) and functionally characterised or validated R. appendiculatus proteins (Bishop et al, 2002;Imamura et al, 2013;Mulenga et al, 2003aMulenga et al, , 2003bPaesen et al, 1999Paesen et al, , 2007Paesen et al, , 2009Preston et al, 2013;Trimnell et al, 2002;Wang and Nuttall, 1995) was performed.…”

“…These four proteins represented 16% of the previously characterised proteins, which was in range with the percent identity observed between the R. appendiculatus transcriptome and the RaGI gene set (15% of best BLAST hits had identities ≤ 90%). No proteins with significant homology were identified for the Japanin-like-RA1, JL-RA1 (Preston et al, 2013) or the Tryptase inhibitor, TdP1 (Paesen et al, 2007) proteins. In order to validate the low identity observed or absence from the dataset of some of the proteins, a subset of 22.4 million reads (about 5% of the sequence reads) was mapped to the nucleotide sequences of the previously characterised R.…”