J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 236 http://jad.tums.ac.ir Published Online: June 30, 2021 Original Article Synergistic Anti-Leishmanial Activities of Morphine and Imiquimod on Leishmania infantum (MCAN/ES/98/LIM-877) Fatemeh Ghaffarifar1; Masoud Foroutan2; *Soheila Molaei3,4; *Eslam Moradi-Asl5 1Department of parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 2Department of parasitology, Abadan Faculty of Medical Sciences, Abadan, Iran 3Zoonoses Research Center, Ardabil University of Medical Sciences, Ardabil, Iran 4Deputy of Research and Technology, Ardabil University of Medical Sciences, Ardabil, Iran 5Department of Public Health, School of Public Health, Ardabil University of Medical Sciences, Ardabil, Iran *Corresponding authors: Dr Soheila Molaie, E-mail: s.molaei@arums.ac.ir, Dr Eslam Moradi-Asl, E-mail: Moradiasl83@yahoo.com (Received 7 Jan 2020; accepted 2 May 2021) Abstract Background: This study was performed to evaluate in vitro and in vivo Leishmanicidal potential of morphine (Mph), imiquimod (IQ), and their combination. Methods: Leishmania infantum promastigote and amastigote assays were performed at the presence of 0.015–150µM Mph, 0.04–416µM IQ, and their combination. The inhibition effects of these drugs on promastigotes were evaluated after 24, 48, and 72h. The cytotoxic effects of the drugs were evaluated by MTT as well as flow cytometry after 72h. We explored the therapeutic effects of Mph and IQ in BALB/c mice at the end of the treatment using parasite load de- termination and cytokine assay. One group of mice received Mph for three weeks before infection. Results: The results of promastigote and amastigote assays showed the cytotoxic effects of the drugs at low concentra- tions. The cytotoxic effects were higher on promastigotes than amastigotes (p< 0.05). There was a negative correlation between drug concentration and amastigote/promastigote viability. Imiquimod alone or combined with Mph showed remarkable cytotoxic effects at all concentrations (p< 0.05). Flow cytometry results revealed apoptosis in the parasite following exposure to the drug combinations. Accordingly, the reduction of parasite loads in the spleen and liver was observed (p< 0.05) with simultaneous increases in IFN-γ and IL-4. We believe that the in vivo leishmanicidal effect was mediated by Mph through IL-4 and by IQ through both IL-4 and IFN-γ. Conclusion: Results pointed out the promising effects of Mph and IQ at low concentrations, especially when combined. Keywords: Morphine; Imiquimod; Synergism effect; Leishmania infantum; Iran Introduction Leishmaniasis is one of the neglected trop- ical diseases, that caused by an obligatory in- tracellular parasite, the genus Leishmania of the family Trypanosomatidae (1). Leishmania parasites are transmitted by Phlebotomus sand flies and their infection leads to cutaneous, mu- cocutaneous, and visceral leishmaniasis depend- ing on parasite species and host immunity (2). Iran is an important focus of cutaneous and visceral Leishmaniasis in the Middle East, in which the visceral form is caused by the L. in- fantum. An epidemiologic study by Mohebali, 2013 showed that the seroprevalence of VL in humans and canines in Iran are 4.7% and 12.2 % respectively (3, 4). The symptomatic form of the disease in humans, is characterized by irreg- ular fever, anemia, hepatosplenomegaly, severe weight loss, globulinemia, and hyperglycemia. The mortality rate is 90–95% in undiagnosed and untreated cases (5). Promastigotes and im- motile amastigotes are the two forms of the parasite. Amastigotes formed in host cells af- Copyright © 2021 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ http://www.arums.ac.ir/file/download/page/1495617715-affiliation.pdf http://www.arums.ac.ir/file/download/page/1495617715-affiliation.pdf mailto:s.molaei@arums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 237 http://jad.tums.ac.ir Published Online: June 30, 2021 ter the bite of ingestion by the female sandfly containing promastigotes (6). Therefore, the parasite avoids a rapid and direct attack by the host immune system and manipulates host- parasite interactions. These results in inhibit- ing the host cell apoptotic machinery and en- sure the parasite’s extended survival in infect- ed cells (7). Different kinds of leishmania spp have been reported to inhibit apoptosis in host cells (8). For example, L. infantum can inhibit drug-induced apoptosis in human macrophag- es (9). Therapeutics employed to treat leish- maniasis are limited and unsatisfactory. They include pentavalent antimony (SbV), ampho- tericin B (AMB) and miltefosine (MF) (10). Despite the inadequate understanding of the mechanism of these drugs, their targets are be- lieved to differ substantially. Antimony, with a rather complex mode of action, has multiple cellular targets. MF disrupts the biosynthesis of glycolipids and glycoproteins as well as the metabolism of alkyl phospholipids. AMB ex- erts its toxic effects since it has a high affinity for the ergosterol of the leishmania plasma membrane (11). However, these agents have some limitations including high cost, long treat- ment duration, route of administration (intra- dermal and intramuscular injection), toxic ef- fects on liver, heart, and kidneys as well as the lack of response to treatment in 10–15% of cases (12). Failure response to antimonial compounds has been reported from endemic areas such as India and also Iran (13). Therapeutics that specifically kill infected macrophages may be beneficial for the treat- ment of leishmaniasis since the parasite resides in host macrophage or monocytes (7). Stim- ulation of opioid receptors in infiltrating cells may be involved in local immune response control or be a signal to produce specific cyto- kines or antibodies analogous to other opioid receptors. Morphine is the main alkaloid in opi- um and an active metabolite of heroin (Bimon- te) (14). Morphine acts through opioid recep- tors (d, l, and j), which is related to Mph-in- duced macrophage apoptosis through reducing the number of murine peritoneal and rabbit alveolar macrophages (15). The Mph-induced apoptosis may be mediated by the up-regula- tion of Bax and p53 proteins, increased p38 MAPK phosphorylation, or TGF-β production by macrophages (16). In vivo and in vitro stud- ies show that Mph inhibits macrophage migra- tion, which is secondary to the apoptotic ef- fects of Mph (17). Imiquimod, a potent toll- like receptor-7 (TLR7) agonist, exerts its ef- fects by the modification of immune respons- es and stimulation of apoptosis (18). Imiquimod modulates immune responses by activating den- dritic cells, macrophages, or other cell types via TLR7, pro-inflammatory cytokines including IFN-a, IFN-c, tumor necrosis factor (TNF) α/β, IL-1a, and IL-12. Treatment with IQ has an im- pact on the expression of various genes in- volved in apoptotic pathways (19). Furthermore, IQ decreases growth and/or increases apoptosis in several human cells (20). Also, IQ has been reported to induce gene ex- pression and protein production of opioid growth factor receptors (21). Currently, extensive re- search is being conducted worldwide to im- prove the treatment strategies of leishmania- sis. The present study was designed to inves- tigate putative promastigote and amastigote in- hibition as well as apoptotic features of Mph, IQ, or their combination on L. infantum and vis- ceral leishmaniasis in BALB/C mice (22-23). Materials and Methods In this study, all applicable international, national and institutional guidelines for the care and use of animals were followed and approved by the Medical ethics committee of Faculty of Medical Science, Tarbiat Modares University and approved under process No. 52D/3593/2015. In vitro Experiments Promastigote and Macrophage Culture Promastigotes of L. infantum reference strain JPCM5 (MCAN/ES/98/LIM-877) provided from http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 238 http://jad.tums.ac.ir Published Online: June 30, 2021 Department of Parasitology, Kerman Univer- sity of Medical Sciences (24). The promastigotes were grown in RPMI-1640 medium (Gibco, dUS) supplemented with 20% heat-inactivat- ed FBS (Gibco, US), 100IU/mL penicillin, and 100μg/mL streptomycin in a humidified atmos- phere at 24 °C. Then, 100µl of medium con- taining 1×106 promastigote cells/mL in loga- rithmic phase were subcultured and seeded in 96-well microplates. Mouse macrophage cells, J774 A1 (CGBR80052901, kindly offered by Professor Marcel Hommel), were cultured in RPMI-1640 medium supplemented with 10% FBS and 100 IU/mL penicillin/streptomycin at 37 °C and 5% CO2 in a humidified atmosphere. Then, they were seeded in 12-well microplates with a density of 1×105 cells/well and used for the next experiments (25-26). Drug preparation Morphine sulfate (Temad Company, Iran) and IQ (Invivogen, Toulouse, France) was pur- chased. Morphine powder was dissolved in 5mL distilled water to obtain a stock solution. One mg/mL stock solution of IQ was prepared by dissolving the powder in a commercially avail- able specific solvent. Then, the stock solutions of both drugs were diluted in RPMI to obtain the concentrations of 0.015–150µM. Glucan- time (273µM) was used as control and pur- chased as a liquid solution (85mg/mL) from Sanofi-Aventis, France (27-28). Study Groups and Treatment of Pro- mastigotes, Macrophages, and Amastigote- infected macrophages Cell treatment with drugs was performed in three groups including L. infantum pro- mastigotes, J774 macrophages, and L. infan- tum promastigote-infected macrophages. In each group, treatments were performed with differ- ent concentrations of Mph, IQ, or Mph +IQ. Parallel exposures to 273µM of Glucantime or culture medium were applied as controls. The treatment groups included (i) Mph (0.015–150 µM), (ii) IQ (0.041–416µM), (iii) Glucantime (273µM), (iv) 0.015–150µM Mph + 0.041– 416µM IQ. Promastigote and Amastigote assay Promastigote assay The promastigotes were treated with dif- ferent concentrations of Mph, IQ, and their combinations for 24, 48, and 72h. Then, the numbers of promastigotes were counted. The percentage of live promastigotes was evaluated by MTT assay after 72h. Promastigotes (1× 106cells/mL) were exposed to studied concen- trations of Mph, IQ, and their combination for up to 72h. Then, the supernatant was removed, and the cells were treated with 5mg/mL MTT ((3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl- tetrazolium bromide, Sigma-Aldrich) solution and incubated for 4h. Afterward, dimethyl- sulfoxide (DMSO) was added, and the absorb- ance was read at 570nm. The results were ex- pressed as the mean percent reduction of par- asite number compared to the untreated con- trols (28). Uninfected macrophage cells viability We evaluated the effects of the drugs on uninfected macrophages, and the percentage of live macrophages was calculated. 1×105cells/ mL of macrophage were cultured and treated with different concentrations of Mph, IQ, and their combinations. The viability of the cells was determined by MTT assay after 72h (28- 29). Amastigote assay After reaching confluency, J774 A1 mac- rophages were seeded on 12-well microplates (Nunc) with a density of 1×105cells/well for 24h. Adherent macrophages were infected with the stationary phase of L. infantum pro- mastigotes at a ratio of 1:10 and were allowed to infect the macrophages for 6h. The cells were washed with fresh RPMI to remove non- phagocytosed promastigotes. Infected macro- phages were further incubated up to 72h in the presence of the drugs. The effects of drugs were http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 239 http://jad.tums.ac.ir Published Online: June 30, 2021 calculated from the percentage of infected cells and the number of amastigotes per infected mac- rophage in treated and untreated cultures in methanol-fixed and Giemsa-stained preparations. Values are means of triplicate exams (29). Evaluation of Apoptosis by flow cytometry Promastigotes, uninfected macrophages, and infected macrophages were treated with dif- ferent concentrations of Mph, IQ, and their combinations. Then, flow cytometry was per- formed to detect apoptotic and necrotic cells using Annexin-V FLUOS Staining Kit (Bio- vision, USA). 1×106 parasites or 1×105 mac- rophages, and infected macrophage cells were treated with the drugs and incubated at 24 °C. After 24h, they were washed with cold PBS and centrifuged at 1500g for 5min. 5μl annex- in-V FITC, 500μL binding buffer and 5μL PI (propidium iodide) were added and incubated for 15 minutes at room temperature. The test was performed using CyFlow® space flow cy- tometry (Sysmex-Partec, USA) and data were analyzed by FloMax software (Partec, version 2.3) (30). All in vitro experiments were per- formed in triplicates. In vivo Experiments Animals and study groups This experimental study was performed at the laboratories of the Parasitology Depart- ment, Faculty of Medical Sciences, Tarbiat Mo- dares University (TMU), Tehran, IRAN from October 2016 to July 2017. Thirty female BALB/C mice, with an average weight of 18– 21g and age of 5–8 weeks, were purchased from Pasteur Institute of Iran. The mice were kept in our animal facility and used for in vi- vo experiments. The mice were randomly di- vided into 5 groups with 5 mice in each group (Table 1). In all the cases, 100μl of pro- mastigotes (2×107cells/mL) at the stationary phase were injected intraperitoneally into the mice. The infected mice were kept for about 18 days to allow parasite growth. Then, the treatments were performed in each group for 3 weeks. 100µl of 1500μM Mph once a week and 100µl of 624μM/mouse IMQ three times a week were injected intraperitoneally. All relevant ethical considerations in animal ex- periments were used before starting the study (Medical Ethics Committee of Tarbiat Mo- dares University, No: 52D/3593/TUMS). Be- fore treatment, two mice were sacrificed and evaluated by parasite culture to confirm the parasite growth. In the 6th group, the mice re- ceived Mph for 3 weeks and were then in- fected with promastigotes. In all groups, after one month, the all of mice were sacrificed. Then, their spleen and liver tissues were eval- uated for parasite load by dilution method. Briefly, 30mg of spleen or liver tissues were homogenized and transferred to RPMI-1640 medium. Five dilutions of the suspensions were prepared, incubated at 26±2 °C, and followed up for two weeks. Finally, the least count of the parasite was considered as the final titer, and parasite load was calculated (26). Extraction of spleen lymphocytes and cyto- kine assay To measure IFN-γ and IL-4 levels, the mice were sacrificed at the end of the treatments, and lymphocytes were isolated from spleen tissue. The staining of lymphocytes was per- formed using trypan blue to obtain the per- centage of live cells. Then, 1×106/mL lym- phocytes were cultured in 12-well plates in RPMI1640 medium. Then, the lymphocytes were stimulated by 20μg/mL of SLA and in- cubated in the presence of 5% CO2 at 37 °C for 72h. Finally, INF-γ and IL-4 levels in cul- ture supernatants were measured by ELISA using U-CyTech kits (bioscience, Netherlands) according to the manufacturer’s instructions. To isolate Soluble Leishmania Antigens (SLA), 108 promastigotes/mL at stationary phase was suspended in PBS and lyzed by five freeze- thaw cycles. The lysates were then centrifuged at 3000g at 4 °C for 15 minutes. The protein concentration of the supernatant was meas- ured using Bradford assay (26). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 240 http://jad.tums.ac.ir Published Online: June 30, 2021 Statistical analysis Data are shown as mean ±SD from at least two independent assays. In in vitro experiments, the differences between the groups were eval- uated by one-way ANOVA. In in vivo, the dif- ferences between treated and control groups were determined by an unpaired Students t-test. P< 0.05 was considered significant by SPSS version 23. Results The results of in vitro experiment Cultured promastigotes were exposed to 0.015–150µM concentrations of Mph, 0.041– 416µM IQ, and their combination. The pro- mastigote inhibition was assayed after 24, 48, and 72h exposure to the drugs as shown in Fig. 1. The results showed that Glucantime, as a positive control, and Mph and IQ in high concentrations had no effect on promastigotes count (p> 0.05). In Mph and IQ group, the con- centrations of a lesser extent had inhibitory ef- fects on promastigotes in all time intervals. This effect was more obvious after 72h (p< 0.05). In the group treated with Mph +IQ, sim- ilar to Mph and IQ only, the concentrations showed the least effects on promastigotes af- ter 72h. The observed promastigote inhibition effect can be attributed to the presence of IQ due to mostly the lack of response in the Mph group. The viability of promastigotes and un- infected macrophages was evaluated after treat- ment with similar concentrations of Mph, IQ, and their combination for 72h (Fig. 2). Pro- mastigote viability results showed a significant effect on the viability for Mph in any doses (p< 0.05) except for 150 and 15µM Mph, 416 and 41.6µM IQ and their combination. The IC50 values of Mph, IQ, and combination for- mula on promastigotes after 72h were 0.102± 0.03, 0.235±0.01, and 0.173±0.02µM, respec- tively. Also, the IC90 values of Mph, IQ, and their combination on promastigotes after 72h were 0.191±0.02, 0.398±0.01, and 0.245±0.02 µM, respectively. The results of the toxic effects of the drugs on uninfected macrophages were similar. Glu- cantime had the most significant effect with no effect of Mph even at high concentrations compared to the control group. In contrast, IQ and the combination of the drugs showed tox- icity on macrophages at higher doses (p< 0.05). The effect of imiquimod on promastigotes in higher doses, whether alone or in combination with Mph, was not significant (p> 0.05). The IC50 values for Mph, IQ, and combination of these drugs were 0.8±0.01, 0.2±0.01, and 0.6± 0.02µM, respectively, after 72h on uninfected macrophages. Fig. 3. Shows the amastigotes count after exposure to all of the studied concentrations of Mph, IQ, and their combination for 72h. In contrast to promastigote assay, glucantime showed a significant effect on amastigotes com- pared to the negative control (p< 0.05). Also, drugs in all doses except 150µM showed sig- nificant toxic effects both on infected macro- phages or intracellular amastigotes compared to the control group (p< 0.05). The same re- sults were observed for IQ alone or its combi- nation with Mph especially at low doses (p< 0.05). Analysis of differences in contaminated macrophages and intracellular amastigotes showed that there were significant differences between the treatment and control groups (p= 0.01) except for high concentrations (Mph 150µM, IQ 416µM, and their same combina- tions). The most significant toxicities were relat- ed to 0.04µM IQ and then 0.015µM Mph com- pared to other groups (p= 0.04). The IC50 val- ues of Mph, IQ, and Mph+IQ on amastigotes after 72h were 18.2±0.3, 0.79±0.1, and 8.72± 0.1µM, respectively. Also, the IC90 values were calculated 29.7±0.1, 1.1±0.1, and 11.8±0.3µM for Mph, IQ, and Mph + IQ respectively. Fig. 4. demonstrates the results of flow cy- tometry analysis. As seen, early apoptosis in promastigote cells was low in morphine treat- ment, 3.04% and 6.08% at 1.5 and 0.015µM, respectively. The necrosis value was 2.55% http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 241 http://jad.tums.ac.ir Published Online: June 30, 2021 and 5.96% at 1.5 and 0.015µM Mph, respec- tively. In contrast, in IQ treatment, early apop- tosis occurred more than necrosis, i.e. 4.11% and 9.18% apoptosis and 3.63%, 2.93% at 4.16µM and 0.04µM of IQ, respectively. When these two drugs were combined, the early apoptosis increased and was 13.72%. The live uninfected macrophage cells were 90.75%, 91.59%, 87.56% at 0.015µM Mph, 0.04µM IQ and 0.015µM Mph+0.04µM IQ, respectively. Apoptosis in infected macrophages was more than in other cells. Significant differences were seen in the values of apoptosis and necrosis in all groups compared with the control (p< 0.05). In vivo experiments Fig. 5. Shows that parasite load in mice was significantly reduced in all drug groups com pared with infected mice treated with no drugs. The highest reduction in parasite load was ob- served in the groups that received 1500Mph treatment before the infection. Also, there was a significant difference between these groups in terms of reduction in parasite load (p< 0.05). As shown in Fig. 6, the levels of both IFN-γ and IL-4 had increased in all treated groups compared to untreated control. In mice treated with Mph, there was a significant re- duction in IFN-γ. Mph seems to inhibit the parasites with a mechanism other than IFN-γ production. As seen, in the Pre-Mph group, the mice were treated before inoculation of para- sites, IFN-γ higher than IL-4 level, but there are no noticeable differences between IL-4 levels compared to the control group (p> 0.05). Table 1. The studied mice groups in the present study (5 mice in each group) Abbreviation Route of Admin- istration Prescription amounts Treatment Mice Groups CTRL(-) - - Uninfected-untreated Control Groups CTRL(+) Intraperitoneal 273μM Infected-untreated (parasite- infected treated with Glucan- time Mph Intraperitoneal 1500μM parasite-infected treated with morphine Treatment Groups IQ Ointment 624μM parasite-infected treated with imiquimod Mph+ IQ Intraperitoneal/ Ointment 1500+624μM parasite-infected treated with morphine+ imiquimod treat- ment Pre-Mph Intraperitoneal 1500μM parasite-infected after pre- treatment with Mph for 3 weeks http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 242 http://jad.tums.ac.ir Published Online: June 30, 2021 Fig. 1. Promastigotes were exposed to 0.015–150µM concentrations of Mph, (0.041–416µM) IQ, and their combination for 24, 48, and 72h. In the negative control group, no treatment was applied. Glucantime was used as a positive control. Data are mean± standard deviation of one experiment in triplicate, *p< 0.05, Tarbiat Modares University, Tehran, 2017 Fig. 2. Viability percentage of promastigotes and macrophages after 72h exposure to 0.015–150µM concentrations of Mph, (0.041–416µM) IQ, and their combination for 72h. After treatment, the viability was evaluated using MTT. In the control group, no treatment was applied. Glucantime was used as a positive control. Data are mean± standard deviations of one experiment in triplicate, *p< 0.05, Tarbiat Modares University, Tehran, 2017 Fig. 3. Amastigotes were exposed to 0.015–150µM concentrations of Mph, (0.041–41µM) IQ, and their combination for 72h. In the control group, no treatment was applied. Glucantime was used as a positive control. Data are mean± SD, *p< 0.05, Tarbiat Modares University, Tehran, 2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 243 http://jad.tums.ac.ir Published Online: June 30, 2021 Fig. 4. The results of flow cytometry analysis of promastigotes, un-infected, and infected macrophages. Early and late apoptosis as well as necrosis has been shown after 72h, Tarbiat Modares University, Tehran, 2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 244 http://jad.tums.ac.ir Published Online: June 30, 2021 Fig. 5. The mice were infected by Leishmania infantum and then treated with studied drugs. At the end of treatment (one week later), the mice were sacrificed, and the parasite loads were evaluated in their liver and spleen. Data were presented as Mean±SD of experiments in 6 mice groups: CTRL (-): infected with no treatment, CTRL (+): parasite- infected with 273μM Glucantime treatment, Mph: parasite-infected with 1500μM Mph treatment, IQ: parasite-infected with 624μM mouse IQ treatment, Mph + IQ: parasite-infected with 1500μM Mph + 273μM IQ treatment, Pre-Mph: parasite-infected after pre-treatment with 1500μM Mph for 3 weeks, Tarbiat Modares University, Tehran, 2017 Fig. 6. The mice were exposed to Leishmania infantum and then treated with studied drugs. Afterward, the mice were sacrificed, and the cytokine levels were evaluated in their spleen using ELISA. Data are presented as Mean±SD devia- tion of experiments in 6 mice groups: CTRL (-): infected with no treatment, CTRL (+): parasite-infected with 273μM Glucantime treatment, Mph: parasite-infected with 1500μM Mph treatment, IQ: parasite-infected with 624μM mouse IQ treatment, Mph + IQ: parasite-infected with 1500μM Mph +273μM IQ treatment, Pre-Mph: parasite-infected after pre-treatment with 1500 μM Mph for 3 weeks, *p< 0.05, Tarbiat Modares University, Tehran, 2017 Discussion Finding new treatments for leishmaniasis has been the target of ongoing efforts for dec- ades. Current medications for the treatment of visceral leishmaniasis are pentavalent antimo- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 245 http://jad.tums.ac.ir Published Online: June 30, 2021 ny, pentamidine, miltefosine, and amphotericin B. However, problems such as the emergence of resistance and severe toxic effects are lim- iting these drugs’ usefulness (31). Thus, sev- eral new drug candidates have been proposed by many researchers (32). In this study, we used Mph and IQ to evaluate their potential toxicity on L. infantum promastigotes as well as uninfected and infected macrophages. The IC50 values of Mph, IQ, and combination for- mula on promastigotes after 72h were 0.102± 0.03, 0.235±0.01, and 0.173±0.02µM, respec- tively. Also, the IC90 values of Mph, IQ, and their combination on promastigotes after 72h were 0.191±0.02, 0.398±0.01, and 0.245±0.02 µM, respectively. These values for uninfected macrophages were 0.8±0.01, 0.2±0.01 and 0.6± 0.02µM, respectively. These data showed that Mph, IQ, and their combination could be con- sidered as new antileishmanial drug candidates at a minimum concentration of drugs. Jabari et al. showed noticeable results of these drugs on Leishmania. major (L. major) in in vitro conditions (28). In our present study, the drugs showed anti-leishmanial effects in the lowest concentration compared to the control group (p< 0.05). On the other hand, simultaneous us- age of Mph and IQ showed promising results against promastigotes compared with the con- trol (p< 0.05). Glucantime, as the positive con- trol, showed a poor effect on promastigotes. Many studies have pointed that glucantime has low anti-leishmanial activities on promastigotes of Leishmania spp (34). The IC50 of Mph, IQ and their combination was found to be 18.2± 0.3, 0.79±0.1, and 8.72±0.1µM, respectively, on L. infantum amastigotes. Also, the IC90 values were calculated 29.7±0.1, 1.1±0.1, and 11.8±0.3µM for Mph, IQ, and Mph +IQ, re- spectively on amastigotes. The low toxicity of these drugs on J774 macrophages indicated the high effect of these drugs on intracellular Leishmania parasites. These results were con- sistent with results obtained by others on L. major amastigotes (27, 33). In the present study, glucantime was more toxic to uninfected and in fected macrophages than Mph and IQ (p< 0. 05). Flow cytometry results indicated a lower value of early and late apoptosis in Mph or IQ alone than their combinations. The results of early and late apoptosis were consistent with other studies that were performed on L. major (28). The difference between our study and oth- ers is perhaps related to drug doses and incu- bation time. The in vivo experiments on BALB/ c mice confirmed the in vitro results. The par- asite burden of spleen and liver tissues in test groups decreased significantly compared to the control group (p< 0.05). The positive syn- ergistic effect of Mph +IQ showed more effi- cacies in controlling the parasite multiplication rate compared to Mph or IQ alone. On the oth- er hand, when Mph was administered before inoculation of parasites, the growth rate of par- asites in BALB/c mice was inhibited, and the parasite rate was at a minimum compared to the control and other test groups. The parasite count decreased to 48%, 63%, 67%, and 80% in Mph, IQ, Mph +IQ, and pre- Mph groups, respec- tively. The results of cytokine assay showed that IL-4 was produced more than IFN-γ in Mph group. In other groups, IFN-γ was pro- duced more than IL-4. The role of Mph as an immunomodulator in protection against leish- maniasis by producing cytokines of CD4 + helper T-cells has been shown in many studies (14). Other similar studies on L. major have shown contradictory results (34). In another study, the augmented effect of Mph at low dos- es was approved in murine visceral leishman- iasis (35). Many studies have shown that these drugs affect the parasite by involving immune system receptors. This is true especially for Mph, which may induce the activation of opi- oid receptors as an immunomodulator (36). Due to the selective pressure of the host defense system, pathogens have evolved different mech- anisms helping them to antagonize apoptotic death of the invaded host cells such as macro- phages. This not only gives more time for par- asite replication (37) but also accelerates the ingestion of apoptotic infected macrophages http://jad.tums.ac.ir/ https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cd4 https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/t-helper-cell J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 246 http://jad.tums.ac.ir Published Online: June 30, 2021 by uninfected ones. This event provides a way for amastigote spreading and subsequent in- fection (38). Promastigotes of L. infantum and their surface lipophosphoglycan have been shown to prevent the apoptosis of macrophag- es, and this allows the survival of intracellular parasites. To unravel the mechanisms behind the resistance of L. infantum to apoptosis, some experiments have been performed. These ex- periments indicate that L. infantum inhibits apop- tosis via the regulation of the IAP (apoptosis inhibitor) family of proteins including cIAP1 and cIAP2. IAP modulation will result in the apoptosis resistance of human macrophages after L. infantum infection. In addition, infec- tion with L. infantum changes the expression of apoptosis-related proteins including Bcl-2, BAX, caspase-3, caspase-8, and caspase-9 (7). This shows that promastigotes employ multi- ple signals to confer more anti-apoptotic effects human macrophages. Although, PI3K is not di- rectly involved in Bcl-2 regulation, a consid- erable level of PI3K phosphorylation occurs in L. infantum-infected cells. In this regard, MAPKs and PI3K are probably engaged dur- ing Leishmania infections and take part in the apoptosis or survival of host cells (39). Opi- oids, including Mph, have immunoregulatory effects and do this through interactions with their receptor on immune cells. They are thus classified as cytokine families. The molecular basis underlying this effect is the modulation of cytokines and altered expression patterns of some cytokine receptors (40). Several research- ers have demonstrated the protective role of Mph against leishmaniasis. In a study, low dos- es of Mph were injected subcutaneously to L. donovani-infected BALB/c mice or hamsters. The results showed significant suppression or even sterile clearing of the infection. In con- trast, high doses exacerbated the infection (41). In another study, Poonawala et al. showed that Mph improved the healing of ischemic wounds through stimulating nitric oxide (NO) via opi- oid receptors (42). The exact mechanisms of the protection role of Mph against leishmania- sis are not completely clear. Macrophages al- so have TLR7, which one of its potent ligands is imidazoquinoline compound IQ (18). It has some roles in the modulation of the immune system through the activation of macrophages or other cells via TLR7 (19). In the present study, Mph and to a lesser extent IQ as well as their combination showed significant tox- icity on promastigotes both in vitro and in vi- vo. In vitro experiments showed that drugs, at high doses, had no effect on promastigotes. The results of Mph were similar to in vivo exper- iments. Mph was not able to induce apoptosis in parasites, but prophylaxis with Mph before infection made the mice resistant to the para- site, an effect that was far more effective than IQ in in vivo. IQ was able to reduce parasite load in vivo, but its effect became stronger when it was synergistically used with Mph. Re- searchers have shown that IQ treatment stimu- lated genes and protein expression of the opi- oid growth factor receptor (21). The synergis- tic effect observed in this study may be at- tributed to this phenomenon. According to the results of many other studies, IQ is effective on Leishmania (43), viruses, and tumor cells (44). Imiquimod is used as a drug in warts, basal cell carcinoma, and Kaposi carcinoma, chronic hep- atitis type C, intraepithelial carcinoma, mela- noma, lung sarcoma, and breast cancer (45). A clinical trial study conducted by Firooz et al. on efficacy and safety of IQ combined with glucantime for cutaneous leishmaniasis of L. tropica expressing no beneficial effect of com- bining treatment with 5% IQ cream with me- glumine antimoniate in patients (46). Another study by Mohebali et al. (47), showed safety, but low efficacy (40.40%) of Alum-ALM mixed with BCG and imiquimod on Canine visceral leishmaniasis. Several studies in Iran and other countries showed contradictory re- sults of imiquimod treatment on CL lesions. The genus of Leishmania, treatment duration, type application of IQ (topical or subcutane- ous injection), and clinical manifestation of le- sions were reported responsible for cure rate in http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 247 http://jad.tums.ac.ir Published Online: June 30, 2021 IQ treatment (46-47). The mechanism of action of IQ is unknown, but it is an agonist of TLR7 and affects immune responses, induce TNFa, IL-1a, and IL-12 pro- duction, and stimulates apoptosis pathways (48). Various cytokines are produced during leish- maniasis such as tumor necrosis factor (TNF) and interferon (IFN), which enhance macrophage activation and other inflammatory responses (49). Macrophages are the main cells against Leishmania and have prominent roles in im- mune response such as phagocytosis. There- fore, to better understand the mechanisms in- volved in killing leishmania, the spleen lym- phocytes were exposed to the parasite. The re- sults showed that in Mph group, increased the level of IL-4 strongly. The results for IQ or IQ +Mph -treated mice were contrasted, showing the equal increase of IL-4 and IFN-γ. This may imply that the apoptosis mechanism is differ- ent in the two treatments. Studies have demon- strated that IQ enhances IFN-γ production both in human and murine cutaneous leishmaniasis. IFN-γ kills the parasite and causes protective immunity. Also, IL-4 enhances programmed apoptosis in stimulated human monocytes (50). Susceptibility to infection is associated with the activation of Th2 or Th1 cells and secre- tion of IL-4, IL-5, IL-6, IL-10, IL- 12, IFN-γ, and lymphotoxins. This could help animals to kill parasites and control infections such as leishmaniasis (51). IFN-γ is important to the immune defense against intracellular pathogens. In leishmaniasis, IFN-γ promotes Th1 differ- entiation and macrophage activity. IFN-γ sig- naling in macrophages results in the activation of host defense mechanisms. Additionally, IFN- γ induces genes such as nitric oxide synthase, the most important molecule responsible for killing leishmania parasites by macrophages (52). Macrophages that are activated by cyto- kines can produce large amounts of nitric ox- ide, one of its functions is a defense against intracellular pathogens particularly Leishma- nia. Nitric oxide has been demonstrated to kill leishmania parasites by inducing amastigotes apoptosis (53). IFN-γ is important since it has immune-stimulatory, immune regulatory, and immune-modulatory effects. In a study on the effects of artemisinin on VL, the authors demon- strated the ability of lymphocytes of infected mice to produce INF-γ during treatment with artemisinin (54). Our study revealed a consid- erable IFN-γ response in the cultured spleno- cyte of test groups. There are several reports on different as- pects of leishmaniasis in the country including the resistant status of vectors to different WHO recommended insecticides, reservoirs, reservoir control, vector control, ecology, novel approach- es, training, and epidemiology (55-90). These re- ports will provide a guideline for disease control. Conclusion According to the results of the present study, Mph and IQ alone or in combination with Mph at low concentrations could inhibit the multi- plication of the L. infantum promastigote and amastigote. The drugs eliminated the parasite growth and the development of murine viscer- al leishmaniasis. Based on current and future in vitro and in vivo studies, Mph and IQ alone or in combination may be considered as a new therapeutic agent for the treatment of visceral leishmaniasis. The authors declare that they have no competing of interests. Acknowledgments The authors wish to gratefully acknowledge Parisa Ebrahimi-Sadr for her technical assis- tance. This work was supported by the depart- ment of parasitology, Faculty of Medical Sci- ences, Tarbiat Modares University of Iran (ap- proval code: NO: 52D/ 1575, Date: 15 March 2015). The authors declare that they have no competing interests. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 248 http://jad.tums.ac.ir Published Online: June 30, 2021 References 1. Goto H, Lindoso JAL (2010) Current diagno- sis and treatment of cutaneous and mu- cocutaneous leishmaniasis. Expert Rev Anti Infect Ther. 8(4): 419–433. 2. Dantas-Torres F (2010) Review of" Human- Animal Medicine-Clinical Approaches to Zoonoses and Other Shared Health Risks" by Peter M. Rabinowitz and Lisa A. Conti (eds.). Parasite Vectors. 3(1): 20. 3. Mohebali M (2013) Visceral leishmaniasis in Iran: Review of the Epidemiological and Clinical Features. Iran J Parasitol. 8 (3): 348–358. 4. Mohebali M, Moradi-Asl E, Rassi Y (2018) Geographic distribution and spatial anal- ysis of Leishmania infantum infection in domestic and wild animal reservoir hosts of zoonotic visceral leishmaniasis in Iran: A systematic review. J Vector Borne Dis. 55(3): 173–180. 5. Barrett MP, Croft SL (2012) Management of trypanosomiasis and leishmaniasis. Br Med Bull. 104(1): 175–196. 6. Moradi-Asl E, Rassi Y, Adham D, Hanafi- Bojd AA, Saghafipour A, Rafizadeh S (2018) Spatial distribution of sand flies (Diptera: Psychodidae; Larroussius group), the vectors of visceral leishmaniasis in Northwest of Iran. Asian Pac J Trop Bi- omed. 8(9): 425–430. 7. Cianciulli A, Porro C, Calvello R, Trotta T, Panaro MA (2018) Resistance to apop- tosis in Leishmania infantum-infected hu- man macrophages: a critical role for anti- apoptotic Bcl-2 protein and cellular IAP1/ 2. Int J Clin Exp Med. 18(2): 251–261. 8. Gupta P, Srivastav S, Saha S, Das PK, Ukil A (2016) Leishmania donovani inhibits macrophage apoptosis and pro-inflam- matory response through AKT-mediated regulation of β-catenin and FOXO-1. Cell Death Dis. 23(11): 1815–1826. 9. Donovan MJ, Maciuba BZ, Mahan CE, McDowell MA (2009) Leishmania in fection inhibits cycloheximide-induced macrophage apoptosis in a strain-depend- ent manner. Exp Parasitol. 12 (1): 58–64. 10. Ouellette M, Drummelsmith J, Papado- poulou B (2004) Leishmaniasis: drugs in the clinic, resistance and new develop- ments. Drug Resist Updat. 7(4–5): 257– 266. 11. Moreira W, Leprohon P, Ouellette M (2011) Tolerance to drug-induced cell death fa- vours the acquisition of multidrug re- sistance in Leishmania. Cell Death Dis. 2(9): e201. 12. Doroodgar M, Delavari M, Doroodgar M, Abbasi A, Taherian AA, Doroodgar A (2016) Tamoxifen induces apoptosis of Leishmania major promastigotes in vitro. Korean J Parasitol. 54(1): 9–14. 13. Erfan MB, Mohebali M, Kazemi-Rad E, Hajjaran H, Edrissian G, Mamishi S, Saffar M, Raoofian R, Heidari M (2013) Downregulation of calcineurin gene is associated with Glucantime® resiatance in Leishmania infantum. Iran J Parasitol. 8(3): 359–366. 14. Alavi NR, Fazaeli A, Pejman B, Ansari H, Fouladi B, Khamesipour A (2009) The efficacy of morphine on murine (BALB/ c) cutaneous leishmaniasis. Iran J Infect Dis Trop Med. 14(46): 15–22. 15. Tubaro E, Borelli G, Croce C, Cavallo G, Santiangeli C (1983) Effect of morphine on resistance to infection. Int J Infect Dis. 148(4): 656–666. 16. Singhal PC, Bhaskaran M, Patel J, Patel K, Kasinath BS, Duraisamy S, Franki N, Reddy K, Kapasi AA (2002) Role of p 38 mitogen-activated protein kinase phos- phorylation and Fas-Fas ligand interaction in morphine-induced macrophage apopto- sis. J Immunol. 168(8): 4025–4033. 17. Malik AA, Radhakrishnan N, Reddy K, Smith AD, Singhal PC (2002) Morphine- induced macrophage apoptosis modulates http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 249 http://jad.tums.ac.ir Published Online: June 30, 2021 migration of macrophages: use of in vitro model of urinary tract infection. J En- dourol. 16(8): 605–610. 18. Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, Horiuchi T, Tomi- zawa H, Takeda K, Akira S (2002) Small anti-viral compounds activate immune cells via the TLR7 MyD88–dependent signaling pathway. Nat Immunol. 3(2): 196–200. 19. Kim SJ, Park GH, Kim D, Lee J, Min H, Wall E, Lee CJ, Simon MI, Lee SJ, Han SK (2011) Analysis of cellular and be- havioral responses to imiquimod reveals a unique itch pathway in transient receptor potential vanilloid 1 (TRPV1)-express- ing neurons. Proc Natl Acad Sci U S A. 108(8): 3371–3376. 20. Urosevic M, Oberholzer PA, Maier T, Hafner J, Laine E, Slade H, Benninghoff B, Burg G, Dummer R (2004) Imiquimod treatment induces expression of opioid growth factor receptor: a novel tumor an- tigen induced by interferon-α? Clin Can- cer Res. 10(15): 4959–4970. 21. Jacob S, Berman B, Nassiri M, Vincek V (2003) Topical application of imiquimod 5% cream to keloids alters expression genes associated with apoptosis. Br J Der- matol. 149(Suppl. 66): 62–65. 22. Honoré S, Garin YJ, Sulahian A, Gangneux JP, Derouin F (1998) Influence of the host and parasite strain in a mouse mod- el of visceral Leishmania infantum infec- tion. FEMS Immunol Med Microbiol. 21 (3): 231–239. 23. Rolão N, Cortes S, Gomes-Pereira S, Campino L (2007) Leishmania infantum: mixed T-helper-1/T-helper-2 immune re- sponse in experimentally infected BALB/ c mice. Exp Parasitol. 11 (3): 270–276. 24. González-De la Fuente S, Peiró-Pastor R, Rastrojo A, Moreno J, Carrasco-Ramiro F, Requena JM, Aguado B (2017) Rese- quencing of the Leishmania infantum (strain JPCM5) genome and de novo as- sembly into 36 contigs. Sci Rep. (1): 1– 10. 25. Dehkordi NM, Ghaffarifar F, Hassan ZM, Heydari FE (2013) In vitro and in vivo studies of anti leishmanial effect of ar- temether on Leishmania infantum. Jun- dishapur J Microbiol. 6(5): e6379. 26. Molaie S, Ghaffarifar F, Dalimi A, Zuhair MH, Sharifi Z (2019) Evaluation of syn- ergistic therapeutic effect of shark carti- lage extract with artemisinin and glucan- time on visceral leishmaniasis in BALB/ c mice. Iran J Basic Med Sci. 22(2): 146– 153. 27. Ebrahimisadr P, Ghaffarifar F, Horton J, Dalimi A, Sharifi Z (2018) Apoptotic ef- fect of morphine, imiquimod and nalmefene on promastigote, infected and uninfected macrophages with amastigote of Leishmania major by flow cytometry. Iran J Pharm Res. 17(3): 986–994. 28. Jabari J, Ghaffarifar F, Horton J, Dalimi A, Sharifi Z (2019) Evaluation of mor- phine with imiquimod as opioid growth factor receptor or nalmefene as opioid blocking drug on leishmaniasis caused by Leishmania major in vitro. Iran J Parasi- tol. 14(3): 394–403. 29. Ebrahimisadr P, Ghaffarifar F, Hassan ZM (2013) In-vitro evaluation of antileish- manial activity and toxicity of arteme- ther with focus on its apoptotic effect. Iran J Pharm Res. 12(4): 903–909. 30. Molaie S, Ghaffarifar F, Hasan ZM, Da- limi A (2019) Enhancement effect of shark cartilage extract on treatment of Leishmania infantum with artemisinin and glucantime and evaluation of killing factors and apoptosis in-vitro condition. Iran J Pharm Res. 18(2): 887–902. 31. Schriefer A, Wilson ME, Carvalho EM (2008) Recent developments leading to- ward a paradigm switch in the diagnostic and therapeutic approach to human leish- maniasis. Curr Opin Infect Dis. 21(5): 483–488. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 250 http://jad.tums.ac.ir Published Online: June 30, 2021 32. Corral MJ, González-Sánchez E, Cuquerella M, Alunda JM (2014) In vitro syner- gistic effect of amphotericin B and al- licin on Leishmania donovani and L. in- fantum. Antimicrob Agents Chemother. 58 (3): 1596–602. 33. Sundar S, Goyal N (2007) Molecular mech- anisms of antimony resistance in Leish- mania. J Med Microbiol. 56(2): 143–153. 34. Ghaffarpasand F, Akbarzadeh A, Heiran HR, Karimi AA, Akbarzadeh A, Ghobadifar MA (2016) Effect of topical Morphine on cutaneous leishmaniasis in an animal model: a preliminary report. Iran Red Cres- cent Med J. 18(5): e24402. 35. Singal P, Singh PP (2005) Leishmania do- novani amastigote component-induced col- ony-stimulating factor production by mac- rophages: modulation by morphine. Mi- crobes Infect. 7(2): 148–156. 36. Lysle DT, Fecho K, Maslonek KA, Dykstra LA (1995) Evidence for the involvement of macrophage-derived nitric oxide in the immunomodulatory effect of morphine and aversive Pavlovian conditioning. The Brain Immune Axis and Substance Abuse. Springer Publishing. North Carolina, US, pp. 141–147. 37. Heussler VT, Küenzi P, Rottenberg S (2001) Inhibition of apoptosis by intracellular pro- tozoan parasites. Int J Parasitol. 31(11): 1166–1176. 38. DaMata JP, Mendes BP, Maciel-Lima K, Menezes CAS, Dutra WO, Sousa LP (2015) Distinct macrophage fates after in vitro infection with different species of Leishmania: induction of apoptosis by Leishmania (Leishmania) amazonensis, but not by Leishmania (Viannia) guyanen- sis. PLoS One. 10(10): e0141196. 39. Ruhland A, Kima PE (2009) Activation of PI3K/Akt signaling has a dominant neg- ative effect on IL-12 production by mac- rophages infected with Leishmania ama- zonensis promastigotes. Exp Parasitol. 122 (1): 28–36. 40. Kardeh S, Ashkani-Esfahani S, Alizadeh AM (2014) Paradoxical action of reac- tive oxygen species in creation and ther- apy of cancer. Eur J Pharmacol. 735: 150– 168. 41. Singh PP, Singal P (2007) Morphine-induced neuroimmunomodulation in murine vis- ceral leishmaniasis: the role (s) of cyto- kines and nitric oxide. J Neuroimmune Pharmacol. 2(4): 338–351. 42. Poonawala T, Levay-Young BK, Hebbel RP, Gupta K (2005) Opioids heal ischem- ic wounds in the rat. Wound Repair Re- gen. 13(2): 165–174. 43. Sacks D, Noben-Trauth N (2002) The im- munology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol. 2(11): 845–858. 44. Oldfield V, Keating GM, Perry CM (2005) Imiquimod. Am J Clin Dermatol. 6(3): 195–200. 45. Geisse J, Caro I, Lindholm J, Golitz L, Stampone P, Owens M (2004) Imiquimod 5% cream for the treatment of superfi- cial basal cell carcinoma: results from two phase III, randomized, vehicle-controlled studies. J Am Acad Dermatol. 50(5): 722– 733. 46. Firooz A, Khamesipour A, Ghoorchi MH, Nassiri-Kashani M, Eskandari SE, Khatami A, Hooshmand B, Gorouhi F, Rashighi-Firoozabadi M, Dowlati Y (2006) Imiquimod in combination with meglumine antimoniate for cutaneous leishmaniasis: a randomized assessor-blind controlled trial. Arch Dermatol. 142(12): 1575–1579. 47. Barati M, Mohebali M, Alimohammadian MH, Khmesipour A, Keshavarz H, Ak- houndi B, Zarei Z (2015) Double-blind randomized efficacy field trial of alum precipitated autoclaved Leishmania ma- jor (Alum-ALM) vaccine mixed with bcg plus imiquimod vs. placebo control group. Iran J Parasitol. 10(3): 351–359. 48. Sauder DN (2000) Immunomodulatory and http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 251 http://jad.tums.ac.ir Published Online: June 30, 2021 pharmacologic properties of imiquimod. J Am Acad Dermatol. 43(1): S6–11. 49. Spellberg B, Edwards Jr JE (2001) Type 1/Type 2 immunity in infectious diseas- es. Clin Infect Dis. 32(1): 76–102. 50. Huang SJ, Hijnen D, Murphy GF, Kupper TS, Calarese AW, Mollet IG (2009) Imiquimod enhances IFN-γ production and effector function of T cells infiltrat- ing human squamous cell carcinomas of the skin. J Invest Dermatol. 129(11): 2676– 2685. 51. Robinson CM, O’Dee D, Hamilton T, Nau GJ (2010) Cytokines involved in inter- feron-γ production by human macrophag- es. J Innate Immun. 2(1): 56–65. 52. Panaro MA, Acquafredda A, Lisi S, Lofru- mento D, Mitolo V, Sisto M, Fasanella A, Trotta T, Bertani F, Consenti B, Bran- donisio O (2001) Nitric oxide production by macrophages of dogs vaccinated with killed Leishmania infantum promastigotes. Comp Immunol Microbiol Infect Dis. 24 (3): 187–195. 53. Holzmuller P, Sereno D, Cavaleyra M, Mangot I, Daulouede S, Vincendeau P, Lemesre JL (2002) Nitric oxide-mediat- ed proteasome-dependent oligonucleoso- mal DNA fragmentation in Leishmania amazonensis amastigotes. Infect Immun. 70(7): 3727–3735. 54. Ghaffarifar F, Heydari FE, Dalimi A, Hassan ZM, Delavari M, Mikaeiloo H (2015) Evaluation of apoptotic and an- tileishmanial activities of Artemisinin on promastigotes and BALB/C mice in- fected with Leishmania major. Iran J Parasitol. 10(2): 258–267. 55. Rassi Y, Jalali M, Vatandoost H (2000) Susceptibility status of Ph. papatasi to DDT in Arsanjan county in Fras Prov- ince, Iran. Iran J Public Health. 29(1–4): 21–23. 56. Rassi Y, Javadian E, Jalali M, Motazedian MH, Vatandoost H (2004) Investigation on zoonotic cutaneous leishmaniasis, south- ern Iran. Iran J Public Health. 33(1): 31– 35. 57. Rassi Y, Javadian E, Amin M, Rafizadeh S, Vatandoost H, Motazedian H (2006) Meriones libycus, the principal reservoir of zoonotic cutaneous leishmaniasis in southern Iran. Eastern Mediterr Health J. 12(3/4): 474–477. 58. Yaghoobi-Ershadi MR, Akhavan AA, Ja- hanifard E, Vatandoost H, Amin Gh, Moosavi L, Zahraei Ramazani AR, Abdoli H, Arandian MH (2006) Repellency ef- fect of Myrtle essential oil and DEET against Phlebotomus paptasi Scopoli, the main vector of zoonotic cutaneous leish- maniasis under laboratory conditions. Iran J Public Health. 35(3): 7–13. 59. Moosa-Kazemi SH, Shayeghi M, Vatan- doost H, Sadeghi MT, Javadian E, Mot- abar M, Hosseini MR, Abtahi M (2009) High performance thin layer chromatog- raphy analysis of deltamethrin residue on the impregnated bed nets during a Leish- maniasis control program in Iran. Iran J Arthropod Borne Dis. 3(1): 1–7. 60. Oshaghi MA, Ravasan NM, Javadian E, Rassi Y, Sadraei J, Enayati AA, Vatan- doost H, Zare Z, Emami SN (2009) Ap- plication of predictive degree day model for field development of sandfly vectors of visceral leishmaniasis in northwest of Iran. J Vector Borne Dis. 46(4): 247–255. 61. Aghaei Afshar A, Rassi Y, Sharifi I, Abai MR, Oshaghi MA, Yaghoobi-Ershadi MR, Vatandoost H (2011) Susceptibility sta- tus of Phlebotomus papatasi and P. ser- genti (Diptera: Psychodidae) to DDT and Deltamethrin in a focus of Cutaneous Leishmaniasis after earthquake strike in Bam, Iran. Iran J Arthropod Borne Dis. 5(2): 32–41. 62. Saeidi Z, Vatandoost H, Akhavan AA, Yaghoobi-Ershadi MR, Rassi Y, Sheikh Z, Arandian MH, Jafari R, Sanei-Dehkordi AR (2012) Baseline susceptibility of a wild strain of Phlebotomus papatasi (Diptera: http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 252 http://jad.tums.ac.ir Published Online: June 30, 2021 Psychodidae) to DDT and pyrethroids in an endemic focus of zoonotic cutaneous leishmaniasis in Iran. Pest Manag SCi. 68(5): 669–675. 63. Veysi A, Vatandoost H, Yaghoobi-Ershadi MR, Arandian MH, Jafari R, Hosseini M, Abdoli H, Rassi Y, Heidari K, Sadjadi A, Fadaei R, Ramazanpour J, Aminian K, Shirzadi MR, Akhavan AA (2012) Com- parative study on the effectiveness of Coumavec and zinc phosphide in con- trolling zoonotic cutaneous leishmaniasis in a hyperendemic focus in central Iran. J Arthropod Borne Dis. 6(1): 18–27. 64. Veysi A, Vatandoost H, Arandian MH, Jafari R, Yaghoobi-Ershadi MR, Rassi Y, Akha- van AA (2013) Laboratory evaluation of a rodenticide-insecticide, Coumavec®, against Rhombomys opimus, the main res- ervoir host of zoonotic cutaneouse leish- maniasis in Iran. J Arthropod Borne Dis. 7(2): 188–193. 65. Saeidi Z, Vatandoost H, Akhavan AA, Yaghoobi-Ershadi MR, Rassi Y, Aran- dian MH, Jafari R (2013) Baseline in- secticide susceptibility data of Phleboto- mus papatasi in Iran. J Vector-Borne Dis. 50: 57–61. 66. Aghai Afshar A, Vatandoost H, Sharifi I, Rassi Y, Abai MR, Oshaghi MA, Yaghoobi-Ershadi MR, Rafizadeh S (2013) First determination of impact and outcome indicators following indoor re- sidual spraying (IRS) with deltamethrin in a new focus of anthroponotic cutane- ous leishmaniasis (ACL) in Iran. Asian Pac J Trop Dis. 3(1): 5–9. 67. Aghai Afshar A, Rassi Y, Sharifi I, Vatan- doost H, Mollaie HR, Oshaghi MA, Abai MR, Rafizadeh S (2014) First report on natural Leishmania infection of Phleboto- mus sergenti due Leishmania tropica by high resolution melting curve method in South-eastern Iran. Asian Pac J Trop Med. 7(2): 93–96. 68. Akhavan AA, Veisi A, Arandian MH, Vatan- doost H, Yaghoobi-Ershadi MR, Hosseini M, Abdoli H, Heidari K, Sadjadi A, Fa- daei R, Ramazanpour J, Aminian K, Shir- zadi MR, Jafari R (2014) Field evaluation of phostoxin and zinc phosphide for the control of zoonotic cutaneous leishman- iasis in a hyperendemic area, central Iran. J Vector- Borne Dis. 51(4): 307–312. 69. Jalilnavaz MR, Abai MR, Vatandoost H, Mohebali M, Akhavan AA, Zarei Z, Ra- fizadeh S, Bakhshi H, Rassi Y (2016) Application of flumethrin pour-on on reservoir dogs and its efficacy against sand flies in endemic focus of visceral leishmaniasis, Meshkinshahr, Iran. J Ar- thropod Borne Dis. 10(1): 78–86. 70. Hazratian T, Vatandoost H, Oshaghi MA, Yaghoobi-Ershadi MR, Fallah E, Ra- fizadeh S, Shirzadi MR, Shayeghi M, Akbarzadeh K, Rassi Y (2016) Diversity of sand flies (Diptera: Psychodidae) in endemic focus of visceral leishmaniasis in Azar shahr district, east Azarbaijan Province, North West of Iran. J Arthro- pod Borne Dis. 10(3): 328–334. 71. Sofizadeh A, Vatandoost H, Rassi Y, Hanafi- Bojd AA, Rafizade S (2016) Spatial Anal- yses of the relation between rodent’s ac- tive burrows and incidence of zoonotic cutaneous leishmaniasis in Golestan Prov- ince, Northeastern of Iran. J Arthropod Borne Dis. 10(4): 569–576. 72. Saghafipour A, Vatandoost H, Zahraei-Ram- azani AR, Yaghoobi-Ershadi MR, Rassi Y, Shirzadi MR, Akhavan AA (2017) Spa- tial distribution of phlebotomine sand flies species (Diptera: Psychodidae) in Qom Province, central Iran. J Med Entoml. 54 (1): 35–43. 73. Saghafipour A, Vatandoost H, Zahraei-Ram- azani AR, Yaghoobi-Ershadi MR, Kara- mi Jooshin M, Rassi Y, Shirzadi MR, Akhavan AA, Hanafi-Bojd AA (2016b) Epidemiological Study on Cutaneous Leishmaniasis in an Endemic Area of http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 253 http://jad.tums.ac.ir Published Online: June 30, 2021 Qom Province, Central Iran. J Arthro- pod Borne Dis. 11(3): 403–413. 74. Sofizadeh A, Rassi Y, Vatandoost H, Hanafi- Bojd AA, Mollalo A, Rafizadeh S, Akha- van AA (2016b) Predicting the distribu- tion of Phlebotomus papatasi (Diptera: Psychodidae), the primary vector of zo- onotic cutaneous leishmaniasis, in Go- lestan Province of Iran using Ecological Niche Modeling: Comparison of MaxEnt and GARP Models. J Med Entomol. 54 (2): 312–320. 75. Veysi A, Vatandoost H, Yaghoobi-Ershadi MR, Jafari R, Arandian MH, Hosseini M, Fadaei R, Ramazanpour J, Heidari K, Sad- jadi A, Shirzadi MR, Akhavan AA (2016) Rodenticide comparative effect of klerat ® and zinc phosphide for controlling zo- onotic cutaneous leishmaniasis in central Iran. Iran J Parasitol. 11(4): 471–479. 76. Saghafipour A, Vatandoost H, Zahraei-Ram- azani AR, Yaghoobi-Ershadi MR, Rassi Y, Jooshin M, Shirzadi MR, Akhavan AA (2017) Control of zoonotic cutaneous leish- maniasis vector, Phlebotomus papatasi, using attractive toxic sugar baits (ATSB). PLoS One. 12(4): e0173558. 77. Shirani-Bidabadi L, Zahraei-Ramazani AR, Yaghoobi-Ershadi MR, Rassi Y, Akhavan AA, Oshaghi MA, Enayati AA, Saeidi Z, Jafari R, Vatandoost H (2017) Assessing the insecticide susceptibility status of field population of Phlebotomus papatasi (Diptera: Psychodidae) in a hyperendemic area of zoonotic cutaneous leishmaniasis in Esfahan Province, central Iran. Acta Trop. 176: 316–322. 78. Arzamani K, Vatandoost H, Rassi Y, Abai MR, Akhavan AA, Alavinia M, Akbar- zadeh K, Mohebali M, Rfizadeh S (2017) Susceptibility status of wild population of Phlebotomus sergenti (Diptera: Psy- chodidae) to different imagicides in an endemic focus of cutaneous leishmania- sis in northeast of Iran. J Vector Borne Dis. 54(3): 282–286. 79. Moradiasl E, Rassi Y, Hanafi-Bojd AA, Vatandoost H, Saghafipour A, Adham D, Aabasgolizadeh N, Omidi Oskouei A, Sadeghi H (2018) The relationship be- tween climatic factors and the prevalence of visceral leishmaniasis in Northwest of Iran. Intern J Pediatrics. 2(50): 7169–7178. 80. Vatandoost H, Nejati J, Saghafipour A, Zahraei-Ramazani A (2018) Geographic and ecological features of phlebotomine sand flies (Diptera: Psychodidae) as leish- maniasis in Central Iran. J Parasitic Dis. 42(1): 43–49. 81. Karimian F, Vatandoost H, Rassi Y, Ma- leki-Ravasan N, Choubdar N, Koosha M, Arzamani K, Moradi-Asl E, Veysi A, Ali- pour H, Shirani M, Oshaghi MA (2018) Wsp-based analysis of Wolbachia strains associated with Phlebotomus papatasi and P. sergenti (Diptera: Psychodidae) main cutaneous leishmaniasis vectors. Pathog Glob Health. 112(3): 152–160. 82. Arzamani K, Vatandoost H, Rassi Y, Akha- van AA, Abai MR, Alavinia M, Akbar- zadeh K, Mohebali M, Rafizadeh S (2018) Richness and diversity of phlebotomine sand flies (Diptera: Psychodidae) in North Khorasan Province, northeast of Iran. J Arthropod Borne Dis. 12(3): 232–239. 83. Karimian F, Vatandoost H, Rassi Y, Ma- leki-Ravasan N, Mohebali M, Shirazi MH, Koosha M, Choubdar N, Oshaghi MA (2019) Aerobic midgut microbiota of sand fly vectors of zoonotic visceral leishman- iasis from northern Iran, a step toward finding potential paratransgenic candidates. Parasite Vectors. 12(1): 1–2. 84. Yaghoobi-Ershadi MR, Akhavan AA, Shir- zadi MR, Rassi Y, Khamesipour A, Hanafi AA, Vatandoost H (2019) Conducting in- ternational diploma course on leishmani- asis and its control in the Islamic Repub- lic of Iran. J Arthropod Borne Dis. 13 (3): 234–242. 85. Rassi Y, Moradi-Asl E, Vatandoost H, Ab- azari M, Saghafipour A (2020) Insecticide http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 236–254 F Ghaffarifar et al.: Synergistic Anti-Leishmanial Activities of … 254 http://jad.tums.ac.ir Published Online: June 30, 2021 susceptibility status of wild population of Phlebotomus kandelakii and Phlebotomus perfiliewi transcaucasicus collected from visceral leishmaniasis endemic foci in northwestern Iran. J Arthropod Borne Dis. 14(3): 277–285. 86. Yousefi S, Zahraei-Ramazani AR, Rassi Y, Vatandoost H, Yaghoobi-Ershadi MR, Af- latoonian MR, Akhavan AA, Aghaei-Af- shar A, Amin M, Paksa A (2020) Evalu- ation of different attractive traps for cap- turing sand flies (Diptera: Psychodidae) in an endemic area of Leishmaniasis, South- east of Iran. J Arthropod Borne Dis. 14 (2): 202–213. 87. Moradi-Asl E, Mohebali M, Rassi Y, Vatan- doost H, Saghafipour A (2020) Environ- mental variables associated with distri- bution of canine visceral leishmaniasis in dogs in Ardabil Province, Northwestern Iran. Iran J Public Health. 49(6): 1033– 1044. 88. Shirani-Bidabadi L, Zahraei-Ramazani AR, Yaghoobi-Ershadi MR, Akhavan AA, Oshaghi MA, Enayati AA, Rassi Y, Gholampour F, Shareghi N, Madreseh E, Vatandoost H (2020) Monitoring of Laboratory Reared of Phlebotomus pa- patasi (Diptera: Psychodidae), main vec- tor of zoonotic cutaneous leishmaniasis to different imagicides in hyper endemic areas, Esfahan Province, Iran. J Arthropod Borne Dis. 14(1): 116–125. 89. Mozaffari E, Vatandoost H, Rassi Y, Mohe- bali M, Akhavan AA, Moradi-Asl E, Za- rei Z, Zahrai-Ramazani A, Ghorbani E (2020) Epidemiology of visceral leishman- iasis with emphasis on the dynamic activ- ity of sand flies in an important endemic focus of disease in Northwestern Iran. J Arthropod Borne Dis. 14(1): 97–105. 90. Rassi Y, Asadollahi H, Abai MR, Kayedi MH, Vatandoost H (2020) Efficiency of two capture methods providing live sand flies and assessment the susceptibility sta- tus of Phlebotomus papatasi (Diptera: Psy- chodidae) in the foci of Cutaneous Leish- maniasis, Lorestan Province, Western Iran. J Arthropod Borne Dis. 14(4): 408–415. http://jad.tums.ac.ir/