Parasitologists United Journal

: 2015  |  Volume : 8  |  Issue : 1  |  Page : 68--77

Immunomodulatory role of ATP inhibitor: glibenclamide and its impact on the pathogenesis of murine Leishmania major infection

Salwa A Shams El-Din 
 Department of Parasitology, Faculty of Medicine, Menoufiya University, Menoufiya, Egypt

Correspondence Address:
Salwa A Shams El-Din
Department of Parasitology, Faculty of Medicine, Shibin El-Kom-YassinAbd El-Ghaffarstreet 32511


Background Leishmania major is the causative agent of cutaneous leishmaniasis in humans. It can also induce visceral or viscerotropic systemic leishmaniasis in humans. Resistance to treatment has been reported in many countries. Glibenclamide (GB) has been found to enhance treatment in resistant cases. Aim The aim of this work was to study the immunomodulatory effect of ATP inhibitor-GB on leishmaniasis caused by L. major. Materials and methods Mice were divided into three groups: group I (GI), noninfected group; group II (GII), infected with L. major; and group III (GIII), infected with L. major and treated with GB, starting 10 days postinfection till the end of the experiment. Evaluation was performed by measuring the size of cutaneous skin lesions, histopathological examination of the liver and spleen, and detection of expression of interferon γ, tumor necrosis factor α, interleukin (IL)-4, and IL-10 cytokines by reverse transcriptase real-time PCR. Transmission electron microscopic study of parasites from peritoneal exudate of GII and GIII mice was also carried out. Results The treated group showed a reduction in skin lesion size, improvement in histopathological manifestations, increased expression of interferon γ, and decreased tumor necrosis factor α, IL-4, and IL-10 expression. Transmission electron microscopic study showed vacuolation and damage of parasites in the treated group. Conclusion GB can be used effectively for the treatment of leishmaniasis.

How to cite this article:
Shams El-Din SA. Immunomodulatory role of ATP inhibitor: glibenclamide and its impact on the pathogenesis of murine Leishmania major infection.Parasitol United J 2015;8:68-77

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Shams El-Din SA. Immunomodulatory role of ATP inhibitor: glibenclamide and its impact on the pathogenesis of murine Leishmania major infection. Parasitol United J [serial online] 2015 [cited 2023 Mar 21 ];8:68-77
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Leishmania are protozoan parasites that reside predominantly in macrophages. This organism causes a spectrum of diseases in humans that is transmitted by infected phlebotomine sandflies. In-vivo, organisms gain entry into phagocytic leukocytes, and transform into an oval, nonmotile amastigote form. Amastigotes replicate intracellularly within macrophage phagolysosomes and spread the infection to adjacent macrophages [1],[2] . Leishmania major is a causative agent of cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) or viscerotropic systemic leishmaniasis in humans [3],[4] , which was considered to be the same as Kala-azar caused by Leishmania donovani [5] . Viscerotropic leishmaniasis is a comparatively mild form of VL [6] . Murine models, such as BALB/c mice, have been shown to be useful experimental models to study the pathogenesis and immunity of Leishmania spp. infection [7] . It was found that the development of a T helper 1 (Th1) response by a resistant host led to the production of interferon γ (IFN-γ) and the development of small lesions with relatively few parasites. However, the inappropriate induction of a Th2 response led to the development of larger lesions with high parasitemia [8] . Meanwhile, interleukin (IL)-10 has been identified as an important mediator of susceptibility in both murine CL and VL [9],[10] . This cytokine has long been shown to be elevated in humans with VL with increased susceptibility to L. major [11] . After Toll-like receptor recognition, most of these pathogens stimulate the production of inflammatory [tumor necrosis factor α (TNF-α) and IL-12] and/or anti-inflammatory (IL-10) cytokines. Also, they prime adaptive immunity by acting on antigen-presenting cells, or directly engage with T cells and trigger a maturation process in antigen-presenting cells, which leads to enhanced production of potent antimicrobial oxidative metabolites and promotes Th1-activated cytokine (IFN-γ) excretion [12],[13],[14],[15] . The expression of IL-12 cytokine is essential for Th1 responses. Deficit in IL-12 levels may underlie the IL-4-dominated response to infection by L. major. TNF-α contributes toward the subsequent dysregulation of macrophages and leads to defective Th1 responses. A low IFN-γ response suggested a defective role in the induction of Th1 response [16] .

Although pentavalent antimonials have been the mainstay of antileishmanial therapy for decades, miltefosine (MIL) has been considered the only oral drug for VL [17] . However, treatment failure rates for both have been reported, where, under antimonial treatment, it increased up to 65% in Bihar between 1980 and 1997, with a marked increase in the failure rate of oral MIL [17],[18] . A significant decrease in MIL efficacy against VL was reported in the Indian subcontinent and the relapse rate was up to 20% within 12 months of treatment [19] . In Nepal, relapse was observed in one-fifth of the MIL-treated patients [20] . A more recent report indicated high MIL treatment failure rates for VL in Nepalese patients. The treatment failure was 21% after 30 days of therapy where each 1-day decrease in MIL exposure was associated with a 1.08-fold increased odds of treatment failure [21] . In Sudanese patients, MIL treatment resulted in 7% relapses among patients with primary VL, of whom 10% developed a new relapse [22] . In addition, widespread antimonial resistance is present in India [23] . The outcome of 2 years of treatment of VL-HIV-coinfected patients with liposomal amphotericin B, followed by combination antiretroviral treatment in Bihar, India, showed frequent VL relapse within 2 years [24] .

Treatment with these drugs is not without complications. In India, pancreatitis is a known side effect of the commonly used drug sodium stibogluconate, and MIL is accompanied by loose motions, vomiting, teratogenicity, and pancreatitis [25] . It was noted that sodium stibogluconate should be avoided for the treatment of VL in HIV-positive patients [26] . Nowadays, amphotericin B treatment failure and relapse rates are particularly high in cases of HIV coinfection, despite initiation of antiretroviral treatment [27] . High-dose amphotericin B is less effective in severely ill HIV-negative patients and HIV-positive patients in Ethiopia [26] .

The enzyme ATP is involved in many physiological processes such as nutrition, osmoregulation, neurobiology, and reproduction of parasites [28] . As pathogenic protozoa rely heavily on the salvage of purine nucleosides from the bloodstream of their host, such compounds are of interest as antiplasmodials and antitrypanosomals [29] . In addition, the ATP-binding cassette (ABC) transporters constitute a family of membrane proteins involved in drug resistance and other biological activities of Leishmania spp. [30] .

The aim of the present study was to study the effect of the ATP inhibitor, glibenclamide (GB), on the pathogenesis of L. major infection in BALB/c mice as an experimental model for human leishmaniasis. Evaluation of the immunological bases of drug response was performed by determination of skin lesion size, histopathological effect, assay of the potential of some cytokines measured by reverse transcriptase (RT) real-time PCR, and transmission electron microscopic (TEM) study on parasites from peritoneal exudates.

 Materials and Methods

Study design

Ninety BALB/c mice were divided into three groups: group I (GI), noninfected control group; group II (GII), infected with L. major intraperitoneally; and group III (GIII), infected with L. major and treated with GB starting 10 days postinfection (PI) till the end of the experiment (10 weeks PI). At 6, 8, and 10 weeks PI, all the measures were done, except for the cytokine assay and TEM, which were done only at 10 weeks PI.


L. major (MRHO/IR/75/ER) amastigotes were maintained by inoculated mice passage, and were used to induce infection in the experimental mice. The strain was obtained from the Parasitology Department, Faculty of Veterinary Medicine, Cairo University (Cairo, Egypt).


Ninety BALB/c mice, 6-8 weeks old, were obtained from the Theodor Bilharz Research Institute. All mice were bred and maintained in the specific pathogen-free animal facilities in the animal house of the Faculty of Medicine, Menoufia University, in accordance with the guidelines for animal research. Mice of GII and GIII were inoculated intraperitoneally with 50 μl of L. major amastigote suspension containing 4 × 10 4 /ml parasites. Mice in control group (GI) (30 mice) were inoculated intraperitoneally with PBS [31],[32] .

The drug

GB, used as an ATP inhibitor, was purchased from EgyPharma (Cairo, Egypt). The dose was adjusted to 80 mg/kg/ml in saline and administered daily orally by an oral cannula. This dose was 40 times lower than the 50% lethal dose (3250 mg/kg) for mice, and was administered 10 days PI till the end of the experiment [33] and before the appearance of skin lesions.

Measurement of skin lesions

The diameter of the resulting lesions in GII and GIII mice was measured in two perpendicular directions and the mean was calculated for each mouse and for the different groups at the different weekly intervals [3] .

Histopathological study

The liver and spleen tissues of each mouse were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin stain [34] .

Cytokine assay by reverse transcriptase real-time polymerase chain reaction

Peripheral blood mononuclear cells (PBMC) were prepared from mice 10 weeks PI from different groups by centrifugation of heparinized blood on Lymphoprep (Nycomed, Oslo, Norway). Gene expression was determined using a Light Cycler Fast Start DNA Master PLUS (Roche Diagnostics, North America, USA) and primers and probes of the TaqMan Gene Expression Assay (Roche Diagnostics, North America, USA) ([Table 1]), and Fast Start DNA Master Hybridization Probes (Roche Diagnostics). mRNA copy number after reverse transcription was calculated using the Light Cycler integrated Quantitative Analysis Program software (version 4.0; Roche Diagnostics). Each experiment was represented as a standard curve of known concentration (10 4 copies/μl). Results were expressed as normalized values of the ratios of cytokine to GADPH and transcription factor to GADPH complementary DNA [42] (Lund et al., 2003).{Table 1}

Total RNA isolation

From the isolated PBMC, total RNA was isolated using a commercially available reagent (TriPure; Roche Diagnostics) following the manufacturer's instructions. Total RNA was treated with 10 U of RQ1 RNase-free DNase (Promega, Madison, Wisconsin, USA), for 30 min, to avoid amplification of contaminating genomic DNA. After the addition of 500 μl of TriPure to inactivate DNase, total RNA was re-extracted. Reverse transcription of mRNA was carried out as follows: 8 μl of water containing 500 ng of total RNA was added to 2 μl of oligo-dT primer (0.5 μg/μl) and incubated at 65°C for 10 min. Samples were chilled on ice and 10 μl of RT mix containing 4 μl 5× RT buffer (250 mmol/l Tris-HCl, pH 8.3, 375 mmol/l KCl, 15 mmol/l MgCl 2 μl deoxynucleotide triphosphate mix 2 (10 mmol/l each), 0.2 μl bovine serum albumin (1 mg/ml), 0.6 μl (25 U) of human placental ribonuclease inhibitor (RNA Guard; Pharmacia Biotech, Gibco Life Technologies, London, UK), 1 μl (200 U) M-MLV RT (Gibco Life Technologies, Scotland, UK), 0.2 μl H 2 O, and 2 μl dithiothreitol (100 nmol/l). The samples were then incubated at 37°C for 60 min [38] .

Polymerase chain reaction on the light cycler with TaqMan probes

The primers used in the present study are presented in [Table 1]. The PCR reaction was carried out in a 20 μl final volume containing H 2 O up to 20 μl, 2 μl two DNA Master Hybridization Probes 10× (DNA Master Hybridization Probes Kit; Roche Diagnostics, North America, USA), 5 μl 25 mmol/l MgCl 2 , 3 μl of 6 pmol/μl forward and reverse primers (final concentration 600 nmol/l), 1 μl of 4 pmol/μl TaqMan probe (final concentration 200 nmol/l), 0.3 μl anti-Taq DNA polymerase antibody (Platinum Taqantibody; Gibco Life Technologies), and 1.0 μl cDNA or standard dilution. After an initial denaturation step at 95°C for 30 s, temperature cycling was initiated. Each cycle consisted of 95°C for 1 s and 60°C for 20 s, the fluorescence being read at the end of this second step (F1/F2 channels, fluorimeter gains regulated on 8 for F1, 2 for F2, and 4 for F3, without color compensation). A total of 45 cycles were performed [38] .

Transmission electron microscopic study

Leishmania parasites from peritoneal exudates of mice in GII and GIII were fixed in a 2.5% glutaraldehyde mixture in PBS, pH 7.4, at 4°C for 24 h and then washed with PBS, postfixed with 1% osmium tetroxide at room temperature, and dehydrated in ascending grades of ethanol. Then, the parasites were placed in acetone, followed by resin : acetone. Finally, the organisms were embedded in resin blocks. Ultrathin sections of 1.0 μm thickness were prepared from the embedded blocks on copper grids. Sections were poststained with uranyl acetate 0.5% and examined by TEM in the EM Unit, Ain Shams University [39] .

Statistical analysis

Data were collected, tabulated, and analyzed statistically by an IBM personal computer and statistical package SPSS (version 13; SPSS Inc., Chicago, Illinois, USA). Two types of statistics were calculated: descriptive statistics [percentage, mean (X), and SD] and analytic statistics (the Mann-Whitney test, Wilcoxon signed-rank test, and Kruskal-Wallis test for non-normally distributed variables) [40] . A P-value of less than 0.05 was considered statistically significant.

Ethical considerations

The study was approved by the Ethical Board of Menoufia University. The animal experimental study was carried out according to the internationally valid guidelines and ethical conditions for the use of animals in the National Research Council (Washington, USA) [41] .


Effect of glibenclamide treatment on leishmanial cutaneous lesions

[Table 2] shows that leishmanial skin ulcer size (lesion size) was reduced from 2.9, 7.4, and 7.8 cm in GII to 1.4, 2.65, and 3.17 cm in GIII at 6, 8, and 10 weeks, respectively ([Figure 1] and [Figure 2]). The reduction in lesion size in GIII versus GII was statistically significant at all tested weekly intervals. No significant difference was detected in lesion size in GII and GIII at all intervals.{Figure 1}{Figure 2}{Table 2}

Histopathological findings

Liver tissue in GII showed a dilated central vein associated with focal inflammatory cells aggregation forming granuloma around the parasite as well as diffuse infiltration in between the degenerated hepatocytes ([Figure 3]). Amastigotes were detected inside the cytoplasm of the macrophages, with diffuse Kupffer cells' proliferation in between the degenerated hepatocytes ([Figure 4]). Liver tissue of mice in GIII showed minimal inflammatory cellular infiltration in the portal area, whereas the hepatocytes showed mild degenerative changes, with no granuloma formation ([Figure 5]). There was lymphoid hyperplasia in the white pulps of the spleen ([Figure 6]). Splenic tissue showed multiple megakaryoblasts in the red pulp ([Figure 7]). {Figure 3}{Figure 4}{Figure 5}{Figure 6}{Figure 7}

Immunological findings

Data in [Table 3] show that the gene expression of IFN-γ was significantly increased in GII and GIII. Also, its expression in GIII was higher than that in GII. However, TNF-α was expressed significantly more in GII than in GIII. The expressions of IL-4 and IL10 expressions compared to GADPH were also significantly reduced in GIII than GII.{Table 3}

Ultrastructural findings

TEM examination of Leishmania parasites in GII showed the parasite inside macrophages as elongated banana shapes with an electron-dense outer membrane, more electron-dense contents, a nucleus, and flagella ([Figure 8], [Figure 9], [Figure 10]). However, in GIII, the parasites showed vacuolation, damage, and loss of flagella ([Figure 11] and [Figure 12]).{Figure 8}{Figure 9}{Figure 10}{Figure 11}{Figure 12}


Both mice and humans infected with L. major parasites may develop VL. Upon infection with L. major, the resistant host develops a dominant Th1 immune response to the parasite antigens, whereas the susceptible one develops a typical Th2 response and succumbs to infection [42],[43] . The immune processes are suppressed by the infection itself, which downregulates the signaling between macrophages and T cells; for example, the production of IFN-γ, and upregulation of nitric oxide (NO) release from IFN-γ-activated macrophages, results in the complete suppression of splenic parasite burden [44] .

The current study aimed to investigate the role of GB as an ATP inhibitor in the treatment of VL. As shown, the GB-treated group (GIII) had reduced cutaneous lesions compared with the nontreated group (GII), confirming the ATP-inhibiting role of GB, with the reduction of parasite physiological and pathological activities in vivo. Similarly, a significant reduction in lesion size was evident when BALB/c mice infected with Leishmania amastigotes were administered GB 20 days after infection [33] . Other recorded drugs also reduced the size of lesions as in acetyl salicylic acid treatment of L. major-infected mice [45] . An intralesional injection of the antitumor cyclopalladated complex in Leishmania-infected BALB/c mice showed a significant decrease in foot lesion size [46] . Also, thiadiazole derivatives significantly decreased lesions in leishmaniasis [47] . Topical treatment with S-nitrosoglutathione of ulcers in BALB/c mice suppressed lesion growth caused by L. major and L. braziliensis [48] . Chemotherapy using pentostam or praziquantel for L. major and Schistosoma mansoni coinfection in BALB/c mice reduced the lesion size [35] . Some plant extracts exerted similar effects on cutaneous lesions as Peganum harmala [36] and Vernonia amygdalina against L. major [49] . Treatment with Pentalinon andrieuxii root extract against L. mexicana [50] and Baccharis uncinella leaves extract against L. amazonensis led to decreased lesion size [51] .

Pathological manifestations of leishmaniasis were reduced in the treated group (GIII), with the disappearance of parasites from the liver and decreased cellular infiltration, congestion, and granuloma formation. Also, in the spleen, there was decreased inflammation and disappearance of parasites compared with the infected group (GII). The upregulation of NO released from IFN-γ-activated macrophages results in the complete suppression of splenic parasite burden [42] . The splenitis found in leishmaniasis is associated with systemic circulation of the infectious agent that induces an increase in the circulation of neutrophils [35] . Similar results were reported with acetyl salicylic acid treatment of L. major-infected mice that inhibited Leishmania visceralization in the spleen and lymph node, and decreased hepatosplenic inflammation by immunomodulation of the NO pathway [45] . Treatment with pentostam and praziquantel resulted in a reduction in changes in the spleen and liver [35] . Granuloma formation around the infected macrophages, which influences healing, occurs by antigen-specific lymphocytes and parasite-specific T cells producing IFN-γ that develop a type 1 response [52],[53],[54] . Treatment of L. major-infected mice with a methanolic extract of V. amygdalina showed attenuation of the histopathological outcome characterized by intact epidermis and less tissue destruction in the skin, spleen, and liver [49] . Thiadiazole derivatives significantly decreased progression of infection in the liver and spleen, and were associated with granuloma formation, which correlates with disease regression in the liver of murine hosts [47] .

The gene expression of IFN-γ at 10 weeks PI was greater in treated GIII than control GI and infected nontreated GII. As recorded previously, infection leads to an increase in IL-10, TNF-β, and IL-4 signals, with a concomitant decrease in the IFN-γ signal. Treatment leads to activation of the correct population of T lymphocytes (Th1) and the production of IFN-γ [55]. Effective primary immunity against L. major in mouse requires IL-12-dependent production of IFN-γ from CD4 + T cells (Th1 response) and CD8 + T cells, which mediates NO-dependent killing by infected macrophages [42],[43] . Cocultured human PBMC with L. major in vitro showed that the principal cytokine produced in GII and GIII was IFN-γ and its production was regulated by IL-10 and IL-12 [56] . Also, TNF-α was reduced in the treated group with minimization of tissue damage and pathological effects. In confirmation, a previous study recorded the greatest lesion sizes in L. mexicana-infected mice expressing TNF-α, but not IFN-γ [57] .

In Peruvian patients with CL, the levels of mRNA for TNF-α, IL-10, and IL-4 were higher and the persistence of high levels of IL-10 in CL was characteristically associated with a poor response to treatment [58] . RT PCR analysis showed upregulation of TNF-α, IL-10, and IL-4 in dermal lesions at the pretreatment stage compared with healthy controls and a significant downregulation after treatment in CL patients [59] . Thiadiazole derivatives significantly stimulated IFN-γ expression and suppressed IL-10 and IL-5 production, favoring type 1 immune responses and resolution of the parasitic infection [47] .

The immunomodulatory properties of garlic in the treatment of leishmaniasis were elucidated in terms of shifting the cytokine response to a Th1 type (IFN-γ) and therefore inducing a protective response [60] . Pentalinon andrieuxii extract also enhanced IFN-γ production in macrophages, in addition to Th1 promotion of the cytokine IL-12 compared with controls [50] . Also, groups of mice treated with Baccharis uncinella leaves extract presented with high amounts of IL-12 and IFN-γ [51] .

In the current study, IL-10 and IL-4 were shown to be reduced in the treated GIII than the infected GII. The inability to resolve infections is associated with the presence of IgG immune complexes that relies on the induction of IL-10 [61] . Susceptibility also correlates with the dominance of an IL-4-driven Th2 response [62],[63] . Protection against L. major corresponded to significant increases in IFN-γ and the low production of IL-4 or IL-10, which suggested an enhanced type 1 response [64] . L. amazonensis-infected lymph nodes showed low expression of IL-4 in the treated group [65] .

The present TEM results of in vivo treatment of Leishmania-infected mice with GB indicated damage of the parasite nucleus in the form of vacuolation and dysregulation of membrane, which were not prominent in nontreated parasites. Similarly, an intralesional injection of antitumor cyclopalladated complexes DPPE 1.2 can destroy L. amazonensis in vitro and in vivo at concentrations that are nontoxic to the host [46] . Electron microscopy of P. andrieuxii extract-treated Leishmania parasites indicated direct membrane damage [50] . Also, topical treatment with S-nitrosoglutathione is cytotoxic to intracellular L. major amastigotes in vitro [48] . The ultrastructural analysis of chloroquine-treated or mefloquine-treated amastigotes showed an accumulation of multivesicular bodies in the cytoplasm of the parasite, suggesting endocytic pathway impairment, in addition to the formation of myelin-like figures [66] .

Trypanosomatid protozoa contain the ATP enzyme in their membranes, and its inhibition by certain drugs was shown to affect the growth and activity of L. amazonensis, Trypanosoma cruzi, and Trypanosoma rangeli [67] . GB was described as a classical inhibitor of the ATP channels in pancreatic cells whose target is the SUR receptor. This receptor is a protein belonging to the ABC transporter family, which has not been identified in Leishmania spp. [68] . ABC transporters have been described for Leishmania spp. [69],[70] . Also, a role for calcium homeostasis seems to be related to the antileishmanial activity of GB [71] . A synergistic effect of GB and IFN-γ on the clearance of L. major by macrophages was reported [71] .

A cause of chemotherapeutic failure in L. major is the active movement of drugs across membranes through ABC transporters. In fact, simultaneous administration of therapeutic drugs with ABC transporter blockers has been invoked to be the means to actively prevent the emergence of drug resistance. GB is a specific blocker of ABC transporters and targets a compartment associated with a multivesicular system that may represent the organelle where drugs are sequestered in L. major parasites [72] . The resistance to amphotericin B was partially reverted by ABC transporters inhibitors in clinical isolates of L. donovani [73] . Inhibition of ATP induced in vitro inhibited cellular growth of Leishmania parasites and also affected the interaction of the parasite with mice peritoneal macrophages [74] . Mitochondria are another site for ATP, and its inhibition in L. donovani induces programmed cell death by significant stimulation of mitochondrial reactive oxygen species production, followed by depolarization of mitochondrial membrane potential. This in turn leads to the oxidative DNA lesions, followed by DNA fragmentation in Leishmania parasites, which could be exploited to develop newer potential therapeutic targets [75] .

In conclusion, from the current study, the cure of leishmaniasis has been shown to depend on a decrease of parasite virulence as well as effective immune response. Both effects were obtained by GB therapy. This indicates that GB may be better as the first line of treatment alone or with antileishmanial drugs for effective results. Also, GB can exert in vivo immunomodulatory activity on immune cells to fight against pathogens.


Conflicts of interest

There are no conflicts of interest.


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