Anti- Toxoplasma gondii effect of Lumefantrine in vitro and in vivo


 Background: Toxoplasma gondii is an obligate intracellular protozoan parasite, which can infect almost all warm-blooded animals, including humans, leading to toxoplasmosis. Currently, the effective treatment for human toxoplasmosis is the combination of sulfadiazine and pyrimethamine. However, both drugs have serious side effects and toxicity in the host. Therefore, there is an urgent need for the discovery of new anti-Toxoplasma drugs with high potency and less or no side-effects. Methods: The cytotoxicity of sulfadiazine and lumefantrine to Vero cells was evaluated by the methyl thiazolyl tetrazolium (MTT) assay. And MTT assay was also used to detect the inhibitory effects of lumefantrine on parasites invasion and proliferation. Flow cytometry was conducted to further verify parasites proliferation. qPCR was performed to evaluate the parasite load in the mice after lumefantrine treatment. In order to determine whether lumefantrine treatment enhances Th1 or Th2 cytokine response, IFN-γ, IL-4, and IL-10 levels in the serum of mice were determined. Results: Our findings suggest that lumefantrine exerts activity against T. gondii by inhibiting its replication and invasion of Vero cells in vitro without being toxic to the cells. Furthermore, lumefantrine protected mice with acute toxoplasmosis from death to a certain extent and reduced the parasite burden in mouse tissues in vivo. In addition, a significant increase in IFN-γ production was observed in high dose lumefantrine-treated mice while IL-10 and IL-4 levels increased in low dose lumefantrine-treated mice. Conclusions: The results of this study demonstrated that lumefantrine may be a promising agent to treat toxoplamosis, and more experiments on the protective mechanism of lumefantrine should be undertaken in further studies.Key words: Toxoplasma gondii, Lumefantrine, anti-Toxoplasma gondii, Invasion, Proliferation


Introduction
Toxoplasma gondii is an obligate intracellular protozoan parasite, which can infect almost all warmblooded, including humans, leading to toxoplasmosis [1,2,3,4,5]. Approximately 30% of the world's population has serological evidence of Toxoplasma infection [6]. T. gondii is a potential threat to both human and animal health. Toxoplasmosis is normally innocuous in individuals with a good immune system, however, T. gondii is quite severe or even fatal for immunocompromised patients, such as those with AIDS, tumour and organ transplant recipients [7][8][9]. In women, primary infection during pregnancy can cause severe damage to fetus and newborns, including blindness, abortion, and stillbirth.
Several anti-T. gondii drugs, including sulphonamides and pyrimethamine to control toxoplasmosis [10]. Both sulphonamides and pyrimethamine prevent the synthesis of folate by inhibiting the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) that are essential for the survival and multiplication of parasites [11,12]. However, these drugs can not completely inactivate encysted bradyzoites or treat congenital toxoplasmosis, and their use is also limited by their side effects, including haematological toxicity (pyrimethamine), cutaneous rash, leukopenia and thrombocytopenia (sulphonamides) [13][14][15][16]. There is increasing evidence of treatment failures in patients affected by toxoplasmosis suggesting the existence of drug resistance in clinical therapy against sulphonamides and pyrimethamine [17].
Continuous efforts have been made to develop drugs for the treatment of toxoplasmosis. However, drug development is an expensive and lengthy process [18]. In an attempt to accelerate the process of drug discovery, older drugs, which are being tested and developed for new activities are making a comeback. Lumefantrine (LF), which was previously named benflumetol, is an antimalarial drug synthesized in 1970s in China [19]. Lumefantrine, which exhibits potent antimalarial activities, with a half-life of 2-4 days, is capable of eliminating the residual parasites that remain in the blood, thereby preventing recrudescence [20].
In Guyana, the combination of lumefantrine and artemisinin has shown a better treatment effect for plasmodium vivax [21]. Plasmodium is an apicomplexa intracellular protozoa, which has similar infection mechanisms to T. gondii. However, the effect of lumefantrine on T. gondii has never been studied. Currently, there is an urgent need for the discovery of new anti-Toxoplasma drugs with high potency and less or no side-effects. Therefore, the aim of this study was to evaluate the activity of Lumefantrine against T. gondii using cell culture and mice infected with T. gondii (RH strain) as in vitro and in vivo experimental models, respectively.

Cytotoxicity assay
The cytotoxicity of sulfadiazine and lumefantrine (Sigma, USA) to Vero cells was evaluated by the methyl thiazolyl tetrazolium (MTT) assay [22,23]. Vero cells (2 × 10 5 ) were seeded in 96-well plates and cultured in 10% FBS DMEM for 12 h to obtain a monolayer. Monolayer cells were washed and directly subjected to lumefantrine (dilution from 94.5 nmol/L to 2.9531 nmol/L) or sulfadiazine(dilution from 500 mg/L to 15.625 mg/L, from 100 mg/L to 3.125 mg/L, from 30 mg/L to 0.9375 mg/L, respectively), which were diluted with 10% FBS DMEM. The cells were subsequently cultured for 24 h

Proliferation assay in vitro
The anti-proliferation effect of lumefantrine on T. gondii was also detected using the MTT assay. Vero cell monolayers in 96-well plates were infected with 1 × 10 6 fresh RH tachyzoites per well and incubated for 2 h. Then, the cell monolayers were washed twice with PBS to remove extracellular tachyzoites and incubated with DMEM (2% FBS) containing different concentrations of lumefantrine (94.5 nmol/L, 17.7188 nmol/L, or 2.9531 nmol/L) for 24 h and 48 h. The sulfadiazine (10 µg/mL) was added as a positive control. Infected cells without drugs were used as a negative control. The MTT assay was carried out to evaluate parasite proliferation as previously described.

Flow cytometry
To further verify parasites proliferation, flow cytometry was conducted. In brief, Toxoplasma-infected cells were digested by trypsin without EDTA after culturing for 24 h, washed twice with PBS, stained with annexin V-FITC (Biolegend, USA) and Propidium Iodide (Biolegend, USA), and incubated at room temperature for 10-15 min without light. Parasite proliferation was measured using a flow cytometer (BD, USA) [24].

Effect of lumefantrine on mice infected by T. gondii
Kunming mice were purchased from Liaoning Changsheng Biotecnology Company, China. Ninety female mice (4-6 weeks, weighing 18-20 g) were divided into 6 treatment groups (15 mice per group). All the mice except the blank control group were infected with fresh T. gondii (100 tachyzoites/mice). After 24 h post-infection, the mice were injected intragastrically with sulfadiazine (10 µg/mL) or lumefantrine(94.5 nmol/L, 17.7188 nmol/L or 2.9531 nmol/L)every two days. Mice in both the blank and parasite control groups were injected intragastrically with the equal amounts of PBS. Mice were observed daily to record the death time and rate. All mice were humanely killed by cervical dislocation to collect blood at 11 days post-infection. Liver, heart, spleen, and lung tissues were collected and stored in liquid nitrogen for RNA extraction.

Real-time quantitative PCR
Tissue RNAs in different groups were extracted using Trizol ((Invitrogen, USA), and the extracted RNAs were treated with DNase I (TaKaRa, China) to completely remove the genomic DNA. The mRNA was reverse transcribed from Oligo (dT) and used as templates for quantitative RT-PCR. Differences were considered statistically significant when P-values were ≤ 0.05.

Cytotoxicity activity
The MTT assay revealed that different concentrations of both lumefantrine and sulfadiazine had no cytotoxicity compared with the negative control cells ( Fig. 1a and 1b). Thus, different concentrations of lumefantrine (high 94.5 nmol/L, medium 17.7188 nmol/L, and low 2.9531 nmol/L) and sulfadiazine (10 µg/mL) were used to carry out further experiments against T. gondii in vitro.

Anti-invasion activity
Vero cell counts using the MTT assay showed that pre-treatment with 94.5 nmol/L, 17.7188 nmol/L and 2.9531 nmol/L lumefantrine reduced tachyzoites invasion by 7.12%, 7.08%, and 6.72% for 18 h, respectively ( Fig. 2A). Sulfadiazine caused a 4.15% reduction after 18 h treatment (Fig. 2A). The invasion ability of pre-treated tachyzoites with different concentrations of lumefantrine and sulfadiazine were significantly reduced compared with the untreated group at 18 h (Toxoplasma group) (P ≤ 0.01).

Anti-Proliferation activity
Further evaluation of the ability of lumefantrine and sulfadiazine to inhibit the intracellular tachyzoite replication within Vero cells was examined using the MTT assay at 24 h and 48 h post-treatment (Fig. 2B). Post-treatment with 94.5 nmol/L, 17.7188 nmol/L or 2.9531 nmol/L lumefantrine reduced the proliferation of tachyzoites by 27.31%, 22.79%, and 20.85% at 24 h and 47.18%, 42.34% and 42.48% at 48 h, respectively (Fig. 2B). A 21.12% reduction at 24 h and 41.2% reduction at 48 h for tachyzoite post-treatment with sulfadiazine were observed (Fig. 2B). This was an indication that lumefantrine could significantly inhibit tachyzoite proliferation compared with the Toxoplasma group (P ≤ 0.01).

Flow cytometry
The anti-proliferation activity of lumefantrine was further examined using flow cytometry. Samples

Parasite load in mice tissues
To further evaluate the parasite load in the mice after lumefantrine treatment, liver, heart, spleen, and lung samples from infected mice were determined by qPCR, and the results are shown in Fig. 5.
Treatment with different concentrations of lumefantrine significantly (**p ≤ 0.01 and *p ≤ 0.05) reduced the parasite load in the liver, heart, spleen and lung tissues compared with the Toxoplasma group (PBS group). The parasite load in different tissues except the liver was also reduced in the positive control group (sulfadiazine group).

Regulation of cytokine levels by lumefantrine in mice infected by T. gondii
In order to determine whether lumefantrine treatment enhances Th1 or Th2 cytokine response, IFN-γ, IL-4, and IL-10 levels in the serum of mice were determined in Fig. 6. Significantly higher levels of IFNγ were observed in mice treated with a high concentration lumefantrine compared to the control groups (p ≤ 0.01). In addition, IL-4 and IL-10 were significantly produced in mice treated with a low concentration lumefantrine compared to the control groups (p ≤ 0.01).

Discussion
Lumefantrine has been shown to have a prominent inhibition effect on P. vivax (sexual and asexual stages) in China [21,25]. Lumefantrine can reduce gametocyte rates in blood and inhibit the development of gametocyte in mosquitoes [21,25]. T. gondii is an apicomplexa intracellular protozoa, which has a similar infection mechanism to Plasmodium. However, studies that have shown successful treatment for toxoplasmosis patients are limited, indicating the urgent need to identify and develop new therapies [26]. In addition, data about the inhibition of T. gondii using lumefantrine is not available. Therefore, in this study, we evaluated the effect of lumefantrine treatment on T. gondii infection in vivo and in vitro .
Cytotoxicity assays showed that lumefantrine was not cytotoxic to Vero cells. Anti-invasion assay showed that the invasion inhibition rate of lumefantrine was about 7% at 18 h post-treatment (p ≥ 0.05), and anti-proliferation assay showed that a 21.12% reduction at 24 h and a 41.2% reduction at 48 h post-treatment with lumefantrine were recorded (p ≤ 0.01). These results indicated that lumefantrine could significantly inhibit the proliferation of T. gondii, which was also verified by Flow cytometry.
Evaluation of anti-T. gondii effects of lumefantrine in mice acutely infected by the RH strain of T. gondii revealed 80%, 66.7%, and 53.3% of mice treated with 94.5 nmol/L, 17.7188 nmol/L, 2.9531 nmol/L lumefantrine, respectively had survived at 11 days post-treatment, and only 46.7% living mice treated with 10 µg/mL sulfadiazine had survived. Furthermore, the parasite burdens in the liver, heart, spleen, and lung after lumefantrine treatment were significantly decreased compared with those in the parasite control group, indicating that lumefantrine exerts an inhibitory effect on T.
gondii, partially provides protection against death due to T. gondii infection, and reduces the parasite

Conclusions
In conclusion, our findings suggest that lumefantrine exerts activity against T. gondii by inhibiting its replication and invasion in vitro in the absence of host toxicity in this study. Furthermore, lumefantrine protected mice with acute toxoplasmosis from death to a certain extent and decreased parasite burden in mice tissues in vivo. Therefore, our results clearly demonstrate that lumefantrine may be a promising agent to treat toxoplamosis in the future, However, more experiments on the protective and therapeutic mechanisms of lumefantrine should be undertaken to fully understand the effects of lumefantrine on Toxoplasma gondii.