Caracterização de receptores Toll-like relacionados à resposta imune no carrapato bovino Rhipicephalus (Boophilus) microplus

Abstract

Toll-like receptors (TLR) are highly conserved transmembrane proteins that play important roles in pathogen recognition. This study aims to identify the presence and differential expression of Toll-like receptors in tissues and cells that are important to the innate immune response of the tick R. (B.) microplus. For this, we identified genes of R. (B.) microplus encoding Toll receptors homologues, which we called RmToll-like and RmToll8-like. We detected the presence of these receptors in organs involved in immune response, such as fat body, hemocytes, salivary glands and digestive cells. Gene expression of Toll-like receptors was evaluated by qPCR and differential expression in the different tissues analyzed. We confirmed the expression of these receptors by western blot and confocal microscopy. Fat body had the greatest modulation of receptors studied, we have chosen to further investigate these receptors in this tissue. In fat body, these receptors were found specifically in round-shaped trophocytes cell membranes and intracellular vesicles. Gene expression of Toll-like receptors was assessed by qPCR 6, 24 and 48 hours after the challenges with the entomopathogenic fungi Beauveria bassiana. Transcription of RmToll-like was positively modulated 48 hours after challenge with B. bassiana, but we did not observe modulation of gene RmToll8-like in the various treatments depending on the time of exposure to the fungi. However, when we evaluated the protein expression of these receptors in ticks challenged with the fungi, no significant difference between treatments and the times analyzed was observed. When we investigated a less virulent strain of fungi we observed that RmToll-like expression was positively modulated after 24h and 48h of infection, this modulation being smaller when compared with those from the more virulent strain. Regarding RmToll8-like we not observed differences between treatments and the times analyzed. The gene expression of antimicrobial peptides was also modulated by the challenge with the fungi, though indicating a possible immunosuppression, but further investigations with new times of challenge should be considered. Evaluating the effects of a pathogen-associated molecular patterns, zymosan, we observed a significant increase in expression of RmToll-like, but not for RmToll8-like. We also evaluated the expression of the corresponding proteins in ticks challenged with zymosan, but no significant difference between treatments and the times analyzed was observed. This study shows for the first time the presence of Toll-like receptors in ticks, and reports the differential expression between different tissues engaged in the innate immune response modulation and immune challenges in this arthropod.