An In Vitro Investigation of the Effects of the Glucocorticoid on Leishmania major Amastigotes

Macrophage Culture and Preparation of the Amastigote- Macrophage In Vitro Model

Subsequently, the J774 mouse macrophage cell line from Pasteur Institute of Iran was used to produce an amastigote-macrophage in vitro model. The cells were isolated from the bottom of the cell culture flask using the cell scraper after massive cell culturing in a RPMI-1640 growth medium enriched with FCS 20%, and penicillin and streptomycin at 37°C and CO2 5%. About 106 cells were transferred to each well with a diameter of 3.5 cm from a 6-well treated cell culture plates containing sterile slides and were incubated at 37°C and CO2 5% for 2 to 3 days after adding 2 mL of RPMI-1640 enriched with FCS 20% complement complex, penicillin, and streptomycin.

The L. major promastigotes were previously counted in the stationary phase and added to each of the wells with a ratio of 7 to 1 (the number of parasites to macrophages). Further, the promastigotes were taken up by macrophages after four hours at 33~34 °C. The extracellular parasites were washed with phosphate-buffered saline and 2 mL of RPMI-1640 enriched growth medium was added to the plates and then the plates were placed at 37 °C for 24 hours to allow them become an amastigote.

Infected Cell Line Treatment With Prednisolone and Mometasone

In the next step, the wells of the cultivating plates were exposed to concentrations of 20, 40, 80, 160, and 320 μg/mL of prednisolone (Sigma p6004) and mometasone (Sigma m4074) as triplicate while three wells without any treatment were considered as the controls. DMSO was used as the solvent for mometasone. More precisely, three wells were exposed to this substance at a concentration of 20 μg/mL to ensure that it was ineffective at the concentration used to dilute the drug. After 24 and 48 hours, the slides were separated from the bottom of the plates and fixed using methanol and stained with Giemsa. In the microscopic investigation step, the number of infected macrophages, as well as the number of intracellular amastigotes was counted, and finally, the average number of amastigotes in each macrophage were calculated by examining 100 macrophages in each drug concentration and compared to the control group. The effect of each treatment on the amastigotes was evaluated using statistical analyses. The supernatant of each well was collected in sterile conditions in small vials and kept for the next steps at -70°C.

Measurement of IL-12 and NO

The amount of IL-12 secreted by the parasite-infected macrophages exposed to different treatments in the supernatant of the culture medium, which was stored at -70°C in the previous step, was measured by the ELISA method using the ELISA kit (Mouse IL-12 Biolegend) according to the kit protocol. Eventually, NO secreted by macrophages in the supernatant of the growth medium when exposed to different concentrations of the drug at 24 and 48 hours was measured by Griess reagent.

Results

The Results of J774 Cell Culturing and Their Infection With Promastigotes

The results showed that 5 to 6 days after culturing J774 cells in 6-well cell culture plates, the cells changed in number, size, and shape (Figure 1a). At this time, promastigotes penetrated into the macrophages and became amastigotes by adding stationary promastigotes to the macrophages and providing other appropriate conditions (Figure 1b).

Figure 1

(a) J774 Cell Line Cultured on 100x Magnification Cell Culture Plates After 6 Days of Culture, (b) Macrophages Containing Phagocytic Parasites Transformed From Promastigote to Amastigote, (c) Macrophages Disintegrated due to High Drug Concentration.

The Results of the Average Numbers of Amastigotes in Infected Macrophages Treated With Drugs

Figures 2 and 3 illustrate the results of the anti-leishmaniasis effect of prednisolone and mometasone by considering the average number of amastigotes in each macrophage.

Figure 2

The Average Number of Amastigotes in Each Macrophage Under Different Concentrations of Mometasone.

Figure 3

The Average Number of Amastigotes in Each Macrophage Under Different Concentrations of Prednisolone

Using the average of the number of amastigotes in the infected macrophages treated with mometasone and prednisolone was significantly different from the control group (P < 0.05). In other words, these drugs reduced the number of amastigotes within the macrophages while the Tukey’s post hoc test showed that the reduction in the number of promastigotes was not time-dependent. Although the effect of the drugs on the parasites was more at concentrations greater than 160 μg/mL, destructive effects were observed in the macrophages, as most of them were decomposed (Figure 1c), therefore, the best concentration was 160 μg/mL for both prednisolone and mometasone.

The Results of IL-12 and NO Secretion From Treated and Untreated Parasitic Macrophages

After the inoculation of macrophages with Leishmania parasite, they were exposed to different drug concentrations and after 24 and 48 hours, the supernatant was collected and the IL-12 and NO secreted by macrophages were measured by the ELISA and Griess reagent methods, respectively. The results demonstrated that IL-12 and NO releases after 24 hours were directly correlated with an increase in the drug concentration and secretion, so that the amount of secreted IL-12 and NO was not significantly different at concentrations less than 160 μg/mL but this difference was significant at higher concentrations.

Although there was a significant difference in the amount of IL-12 and NO secreted by different drug concentrations at 24 hours, the results of Tukey’s post hoc test showed that this was not time-dependent and at 48 hours after adding the drug, the amount of IL-12 and NO secreted represented no differences compared to the control group.

The results of the mean concentration of IL-12 and NO secreted by macrophages under different concentrations of the drug were shown in Figures 4 and 5.

Figure 4

NO Secretion by Macrophages Infected With Leishmania Parasite Under Different Concentrations of Mometasone and Prednisolone.

Figure 5

The Secretion of IL-12 by Macrophages Infected With Leishmania Parasite Under Different Concentrations of Mometasone and Prednisolone.

Discussion

The chemical treatment of leishmaniasis heavily relies on the development of the immune response that activates macrophages to produce toxic nitrogen and oxygen mediators to kill amastigotes. This process is suppressed by the parasite itself, which results in modest signaling between the macrophages and the T-cells. During the treatment of macrophages with the glucocorticoid, the genes responsible for inducing NO synthase production increase leading to the production of a large amount of NO (1).

On the other hand, the main mechanisms of the macrophage cells are the destruction of amastigotes, the production of cytokines such as IL-12, NO, and toxic oxidizing metabolites such as superoxide and hydrogen peroxide, which are harmful to all stages of the parasite (14).

In the present study, it was found that the average number of amastigotes in the infected macrophages treated with mometasone and prednisolone was significantly lower than that of the control group and these drugs reduced the number of amastigotes within the macrophages. Moreover, the best concentration of both drugs was 160 μg/mL due to the destructive effects on macrophages and their degradation at high doses. Osero et al studied the effect of glucocorticoids (hydrocortisone and dexamethasone) on macrophages infected with the L. major parasite and found that the parasite in the macrophages of the treated group with glucocorticoids reduced compared to the control group. They further suggested glucocorticoids to control leishmaniasis infection (1). These findings are in line with those of the present study.

The parasites of the genus Leishmania are capable of inhibiting the effector functions of macrophages. These parasites develop the adaptive ability to escape the host defenses. For example, they inactivate the NF- κ B complex and suppress iNOS expression in infected macrophages, which are responsible for the production of the major antileishmanial substance NO, favoring then its replication and successful infection.

According to some studies, the inhibition of the NO production led to the disability of macrophages for inhibiting L. major reproduction in the culture medium (17,18). In this study, NO production was observed by the macrophage stimulated with prednisolone and mometasone. NO is a potent microbial agent that causes the death of intracellular parasites and other pathogens (19-22). Another study reported the need for iNOS for the response of NK cells to L. major (14).

Recently, several studies have shown that NO production in human monocytes or macrophages in vitro is also stimulating (16,23). IFN-γ and INF-α have synergistic effects on the induction of NO and iNOS by macrophages in vitro (24).

The results of this study indicated that the secreted NO had a significant increase by increasing the concentration of the drugs after 24 hours. The results of another study showed similar results, demonstrating that glucocorticoids increased the expression of TNF-α genes, along with NO and IL-1 in macrophages as compared to the control group (1).

IL-12 increases cellular immunity by enhancing the differentiation of TCD4+ cells into the Th1. Additionally, the role of IL-12 in the activation of Nk cells leads to the activation of macrophages because it increases the production of IFN-γ from NK cells, as well as IL-12 and IL-18, has a greater effect on the stimulation of IFN-γ production by Nk cells (25-27).

Finally, cytokines that increase the response of Th1 such as IL-12 are more secreted and IL-12 is an essential stimulant for secreting IFNγ and inducing intrinsic and cellular responses against intracellular parasites including Leishmania and IL-10 acts in a modest way for these reactions (28).

In this study, IL-12 secreted by macrophages was measured and the results represented that the amount of IL-12 secretion was significantly and positively correlated with the increase in drug concentration after 24 hours (P < 0.05).

Based on the results of other studies, human cytokine response to L. major at the initial stage of infection and in patients susceptible to treatment increased IL-12 and IFN-γ expression, but lower levels of Th2 cytokines were secreted as IL-5 and IL-4 (29-30).

The results of the present study showed that the average of L. major parasites in mouse J774 cells decreased with prednisolone and mometasone. In addition, the level of NO and the mean level of IL-12 secreted by infected macrophages treated with both drugs indicated an increase. The decrease in amastigotes appears to be due to an increase in the mean level of NO and IL-12.

Although glucocorticoids in this study and previous studies did not completely eliminate the parasite, in vitro experiments reduced parasites while increasing cytokines in the macrophages (1). On the other hand, the symptoms of the disease, the size of the lesion, and the duration of the disease reduced in in vivo experiments (31). Therefore, the use of glucocorticoid drugs has many benefits. Glucocorticoids are available and considered as cost-effective drugs. Further, their efficiency is approved regarding reducing the symptoms in the case of many inflammatory diseases. Furthermore, their mechanism in the inhibition of the NF-kβ transcription factor, which increases the level of Th1 cytokines that are effective in treating the leishmaniasis lesion. Accordingly, glucocorticoids can probably be useful in combination with anti-leishmaniasis drugs for reducing the parasitic infections and managing immunity toward Th1 and faster treatment with fewer side effects.

Conclusions

In this study NO and mean IL-12 secretion were measured as major macrophage cytokines against L. major in order to evaluate the performance of glucocorticoid drugs. The results of the analysis showed that the amount of NO and mean IL-12 in the supernatant in treated groups with prednisolone and mometasone drugs increased compared to the control groups. Given the addition of drugs without any inducer, glucocorticoid drugs may directly affect the function of macrophages and increase the release of NO and the mean IL-12. Thus, it is recommended to investigate the effect of glucocorticoids on leishmaniasis in vivo in order to obtain more information.

Acknowledgements

This article is a master’s degree program in parasitology under number 397049 in collaboration with Isfahan University of Medical Sciences. We appreciate the collaboration of the Research Deputy and the Department of Parasitology of Isfahan University of Medical Sciences.

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