Evaluation of the in vitro expression of ATP binding-cassette (ABC) proteins in an Ixodes ricinus cell line exposed to ivermectin
© Mangia et al. 2016
Received: 8 January 2016
Accepted: 5 April 2016
Published: 18 April 2016
Ticks are among the most important vectors of pathogens causing human and animal disease. Acaricides are used to control tick infestation, although there are increasing reports of resistance. Recently, over-expression of ATP-binding cassette (ABC) transporter proteins (P-glycoproteins, PgP) has been implicated in resistance to the acaricide ivermectin in the ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus sanguineus sensu lato. Ixodid tick cell lines have been used to investigate drug resistance mechanisms. The aim of the present study was to evaluate expression of several PgPs in the Ixodes ricinus-derived cell line IRE/CTVM19 and to determine modulation of expression following treatment with ivermectin.
IRE/CTVM19 cells were treated with different concentrations of ivermectin (0, 11, 22 or 33 μM) and incubated for 10 days. Evaluation of viability and relative expression of ABCB1, ABCB6, ABCB8 and ABCB10 genes were carried out at day 10 post treatment. Cell viability ranged between 84 % and 92 % with no significant differences between untreated and treated cells. qRT-PCR showed that ABC pump expression was not significantly modulated by ivermectin treatment. Expression of the ABCB8 PgP subfamily revealed a biphasic trend, based on the ivermectin concentration. ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected.
This is the first report of PgP expression in an I. ricinus-derived tick cell line. Development of an in vitro model for the study of acaricide resistance mechanisms would greatly facilitate screening for drug resistance in ticks.
Ticks are among the most important vectors of a wide range of pathogens causing human and animal diseases, and several classes of acaricide are widely used to control tick infestation [1, 2]. However, there is an increasing number of reports of resistance to acaricides including macrocyclic lactones . One of the most widely studied mechanisms of drug resistance is associated with the protein family of ABC transporters, which transport toxic substances outside the cell, thereby reducing their concentration inside the cell . These efflux pumps are able to eliminate both endogenous and exogenous toxins and are an important “first-line” defence mechanism. Recent studies have shown that ABC transporters are present in a wide range of organisms, including mammals and arthropods, and have been implicated in drug resistance in ticks [5–7]. A recent study reported that over-expression of a gene encoding for ABC-multidrug transporters was associated with in vitro-induced resistance to ivermectin in the tick cell line BME26 , derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus . In addition, ABC transporters may be involved in detoxification in the brown dog tick Rhipicephalus sanguineus (sensu lato) . Despite increasing evidence that ABC transporters are likely to be involved in acaricide resistance in ticks, there have been no studies in Ixodes ricinus, one of the most important vectors of pathogens causing tick-borne diseases in Europe. Ixodid tick cell lines have already been used as a model for the study of drug resistance [8, 11]. The development of an in vitro model for the study of molecular resistance mechanisms and the screening of potential genetic markers of resistance in I. ricinus would be of great scientific interest.
The aim of the present study was to evaluate the expression of selected members of the ABC transporters subfamily B (ABCB1, ABCB6, ABCB8 and ABCB10) in vitro, following ivermectin treatment of the I. ricinus cell line IRE/CTVM19. Ivermectin was chosen as the test acaricide both because of its use in previous published studies on ABC transporters [6–8, 10] and because it has been shown to be active against I. ricinus ticks .
All reagents were purchased from Sigma Aldrich (Milan, Italy) except where indicated.
Cell line maintenance
Cells of the I. ricinus embryo-derived cell line IRE/CTVM19  were seeded in flat sided 10 ml tubes (Nunc) in Leibovitz’s L-15 medium (Life Technologies, Milan, Italy) supplemented with 20 % fetal bovine serum, 10 % tryptose phosphate broth, 2mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml) and incubated at 28 °C. Medium (3/4 volume) was replaced weekly and cells were split at intervals of at least 15 days.
Treatment of IRE/CTVM19 cells with ivermectin
IRE/CTVM19 cells seeded at a concentration of 3 × 106 cells/ml in 2 ml culture medium per tube were treated immediately with different concentrations of ivermectin in 0.1 % DMSO (11 μM, 22 μM or 33 μM). Untreated cells and cells treated with 0.1 % DMSO only served as controls. Cultures were incubated for 10 days and medium was changed on the seventh day. Replacement media contained the same concentrations of ivermectin as reported above.
Growth curve and cell viability
For growth rate analysis, four replicate tubes were used per treatment. On days 0, 5 and 10, a small aliquot of cell suspension was harvested from each tube, labelled with Trypan Blue 0.4 % w/v and counted using a haemocytometer. A test of cell viability was also performed on day 5 of treatment using the LIVE/DEAD Fixable Near-IR stain kit (Life Technologies). Cells were stained according to the manufacturer’s instructions and analysed by flow cytometry. Flow cytometry was performed using a BD FACSVerse (BD Biosciences, Stockholm, Sweden) equipped with 488 nm blue and 633 nm red lasers, and results were analysed using the FACSDiva (BD Biosciences) software. Cells frozen at -80 °C and thawed three times were used as negative controls.
RNA extraction and determination of gene expression profile after acaricide treatment
Primers used in the present study for evaluation of expression of the ABC subfamily B genes (ABCB1, ABCB6, ABCB8 and ABCB10) in the Ixodes ricinus cell line IRE/CTVM19
ABC-B subfamily protein expression was then evaluated by quantitative RT-PCR, using the SYBR Green master mix kit (EuroClone), according to the manufacturer’s instructions. The final concentration of each primer in all the reactions was 0.4 μM. The amplification protocol was characterised by a denaturation step (95 °C for 2 min) and 45 repeated cycles (95 °C for 10 s; 56 °C for 15 s; 72 °C for 20 s). Fluorescence signals were collected in every cycle and the presence of nonspecific products was excluded through analysis of the melting curves. Results were presented as the mean ± S.E.M. of three experiments with four replicates each, managed by CFX Manager software (Biorad) and expressed as Relative Normalised Expression (ΔΔCq).
One-way ANOVA with Dunnett’s post-hoc test was performed using GraphPad Prism version 6(GraphPad Software, San Diego California USA, www.graphpad.com). P-values < 0.05 were considered statistically significant.
Results and discussion
To the authors’ knowledge, this is the first report of expression and modulation of ABC transporters in an I. ricinus-derived cell line. The relative expression of ABC genes in ivermectin-treated cells ranged between 0.5 and 1.6 fold compared to the time zero control. Interestingly, ABCB8 expression showed a particular, biphasic dose-response relationship, with a low-dose stimulation and a high-dose return to the control level, as reported by Calabrese for drug-resistant vertebrate cell lines . The results presented here indicate the need for further study.
Furthermore, ivermectin has been incriminated as an inhibitor of detoxification mechanisms in mammalian cell lines  and the results regarding ABCB8 in the present study suggest that the inhibitor effect may also be true for I. ricinus tick cell lines. In the only other similar in vitro study published so far , a clear role for ivermectin could be demonstrated both in terms of establishment of a lethal concentration 50 (LC50) and of a resistant tick cell sub-line. In that study, up-regulation of several ABC genes (ABCB10, ABCC1, ABCB7, ABCC2) was observed in the ivermectin-resistant cell sub-line BME26-IVM.
Interestingly, the I. ricinus cell line used in the present study was able to tolerate a much higher concentration of ivermectin, 30 μg/ml (33 μM), than the unselected BME26 cell line that did not survive after exposure to a concentration of ivermectin of 12.5 μg/ml or the resistant sub-line BME26-IVM for which the LC50 was calculated as 15.1 μg/ml . Differences between the biology of the two tick species from which the cell lines were derived (I. ricinus and R. microplus), between the phenotypic composition of the cell lines themselves and/or in the culture conditions used (such as medium composition and incubation temperature) may explain the differences in the outcome of treatment and should be taken into account when refining the in vitro model of I. ricinus. Finally, it would be of interest to develop this in vitro model with cell lines from other economically important tick species and to evaluate the effect of different acaricides that have been reported to be losing their efficacy in the field .
The present study was funded by PRIN project number #2010PJJ4W3_004. The tick cell line IRE/CTVM19 was provided by the Tick Cell Biobank at The Pirbright Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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- Dantas-Torres F, Chomel BB, Otranto D. Ticks and tick-borne diseases: a One Health perspective. Trends Parasitol. 2012;28(10):437–46.View ArticlePubMedGoogle Scholar
- Dantas-Torres F, Otranto D. Best practices for preventing vector-borne diseases in dogs and humans. Trends Parasitol. 2015;32(1):43–55.Google Scholar
- Otranto D, Wall R. New strategies for the control of arthropod vectors of disease in dogs and cats. Med Vet Entomol. 2008;22(4):291–302.View ArticlePubMedGoogle Scholar
- Krishna R, Mayer LD. Multidrug resistance (MDR) in cancer. Mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs. Eur J Pharm Sci. 2000;11(4):265–83.View ArticlePubMedGoogle Scholar
- Lara FA, Pohl PC, Gandara AC, Ferreira Jda S, Nascimento-Silva MC, Bechara GH, Sorgine MH, Almeida IC, Vaz Ida Jr S, Oliveira PL. ATP binding cassette transporter mediates both heme and pesticide detoxification in tick midgut cells. PLoS One. 2015;10(8):e0134779.View ArticlePubMedPubMed CentralGoogle Scholar
- Pohl PC, Klafke GM, Carvalho DD, Martins JR, Daffre S, da Silva Vaz Jr I, Masuda A. ABC transporter efflux pumps: a defense mechanism against ivermectin in Rhipicephalus (Boophilus) microplus. Int J Parasitol. 2011;41(13–14):1323–33.View ArticlePubMedGoogle Scholar
- Pohl PC, Klafke GM, Junior JR, Martins JR, da Silva Vaz Jr I, Masuda A. ABC transporters as a multidrug detoxification mechanism in Rhipicephalus (Boophilus) microplus. Parasitol Res. 2012;111(6):2345–51.View ArticlePubMedGoogle Scholar
- Pohl PC, Carvalho DD, Daffre S, Vaz Ida Jr S, Masuda A. In vitro establishment of ivermectin-resistant Rhipicephalus microplus cell line and the contribution of ABC transporters on the resistance mechanism. Vet Parasitol. 2014;204(3–4):316–22.View ArticlePubMedGoogle Scholar
- Kurtti TJ, Munderloh UG, Ahlstrand GG, Johnson RC. Borrelia burgdorferi in tick cell culture: growth and cellular adherence. J Med Entomol. 1988;25(4):256–61.View ArticlePubMedGoogle Scholar
- Cafarchia C, Iatta R, Immediato D, Puttilli MR, Otranto D. Azole susceptibility of Malassezia pachydermatis and Malassezia furfur and tentative epidemiological cut-off values. Med Mycol. 2015;53(7):743–8.View ArticlePubMedGoogle Scholar
- Cossio-Bayugar R, Wagner GG, Holman PJ. In vitro generation of organophosphate resistant Boophilus microplus (Acari: Ixodidae) cell lines. J Med Entomol. 2002;39(2):278–84.Google Scholar
- Taylor SM, Kenny J. An ivermectin sustained release bolus in cattle: its effects on the tick Ixodes ricinus. Med Vet Entomol. 1990;4(2):147–50.View ArticlePubMedGoogle Scholar
- Bell-Sakyi L, Zweygarth E, Blouin EF, Gould EA, Jongejan F. Tick cell lines: tools for tick and tick-borne disease research. Trends Parasitol. 2007;23(9):450–7.View ArticlePubMedGoogle Scholar
- Lawson D, Arensburger P, Atkinson P, Besansky NJ, Bruggner RV, Butler R, Campbell KS, Christophides GK, Christley S, Dialynas E, et al. VectorBase: a data resource for invertebrate vector genomics. Nucleic Acids Res. 2009;37(Database issue):D583–587.View ArticlePubMedPubMed CentralGoogle Scholar
- Calabrese EJ. P-glycoprotein efflux transporter activity often displays biphasic dose-response relationships. Crit Rev Toxicol. 2008;38(5):473–87.View ArticlePubMedGoogle Scholar
- Ballent M, Lifschitz A, Virkel G, Sallovitz J, Mate L, Lanusse C. In vivo and ex vivo assessment of the interaction between ivermectin and danofloxacin in sheep. Vet J. 2012;192(3):422–7.View ArticlePubMedGoogle Scholar
- Eiden AL, Kaufman PE, Allan SA, Oi F. Establishing the discriminating concentration for permethrin and fipronil resistance in Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae), the brown dog tick. Pest Manag Sci. 2015;32(1):43-55.Google Scholar