Cutaneous leishmaniasis (CL) is a self-healing skin disease which rarely for unknown reason(s) the lesion develops to a non-healing form. It seems that the initial contact of Leishmania parasites with the host innate immune system is an important step in the outcome of the disease. Recent studies suggested that toll-like receptors (TLRs) play a role in Leishmania recognition. In this study, the level of TLR2 and TLR4 was checked in patients with healing form of lesion and compared with that of patients with non-healing form of lesion caused by Leishmania major. Gene expression of TLR2 and TLR4 in peripheral blood-derived macrophages, before and after stimulation with live L. major promastigotes, was evaluated using quantitative real-time reverse transcription PCR and flow cytometry. The results showed that the mean relative gene expression and difference membrane expression of TLR2 in macrophages of patients with healing form of lesion were significantly higher than patients with non-healing form of lesion (P < 0.0001 and P = 0.0034), respectively, and the mean relative gene expression and difference in protein expression of TLR4 in macrophages of patients with healing form of lesion were significantly higher than that of patients with non-healing form of lesion (P = 0.021 and P = 0.002), respectively. The data suggested a possible role for TLR2 and TLR4 in the outcome of CL lesion. Further studies are needed to understand more about the detail role of the immune factors in leishmaniasis.
Leishmaniasis is a complex disease caused by various species of Leishmania with a wide range of clinical manifestations and still is a major public health problem in some endemic countries . Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis [2, 3]. Although CL is a self-healing disease but rarely develops to a non-healing form of lesion which does not respond to current available therapies [4-6], clinical manifestations of leishmaniasis depend upon the species of Leishmania and the host genetic makeup governs the type of immune response generated . Acquired immune responses in leishmaniasis are extensively studied in animal model and human [8-15], but there are a few studies on the role of innate immune response in leishmaniasis [16-19]. There are plenty of publications on innate immune response in animal model of leishmaniasis, but only a few studies are completed in human leishmaniasis and still the role of innate immune response in human leishmaniasis is not clear [17, 18, 20]. Monocytes migrate to the site of infection and differentiate into macrophages, which participate in phagocytosis . At the initial phase of infection, the ability of Leishmania species to invade and survive inside macrophages depends upon the interaction between Leishmania and macrophage surface molecules . The early events during interaction of host cells with the parasite and production of inflammatory mediators play crucial role in the outcome of the disease . The pattern recognition receptors (PRRs) are expressed on the phagocytosis, which involve in the detection of pathogen-associated molecular (PAMPs) on the surface of the pathogens [24, 25]. Toll-like receptors (TLRs) are well-characterized class of PRRs , so far 13 types of TLRs are described and every one of them recognizes distinct molecules of micro-organisms . TLRs bind to myeloid differentiation factor 88 (MyD88), a protein that interacts with several other molecules in a signalling cascade that leads to cytokines production . The activation of macrophage functions is first mediated by Toll-like receptors (TLRs), which play an important role in the control of infection . TLRs play an essential role in linking the innate and adaptive immunity and enhance phagocytosis and killing process of the parasites . TLR recognition is often associated with the production of proinflammatory cytokines and generation of other effector molecules, which promote differentiation of Th1 cells, and it is important to know about TLR activation in Leishmania infections . There are limited studies on TLR2 and TLR4 in animal model and possible relation to Leishmania infections. Activation of TLR2 and TLR4 molecules in human leishmaniasis and the outcome of the diseases are not well known [31-34].
Control measures in leishmaniasis are not fully effective, and inspite of ample evidence showing that the development of an effective vaccine is possible, yet there is no vaccine available against any form of leishmaniasis known [35, 36], partly due to the lack of enough information on the immunopathology surrogate markers of leishmaniasis that justifies studies on immune responses and the factors involved in disease control, particularly the innate immunity and the initial steps of interaction between parasite and the host immune cells. Available chemotherapy is not fully effective, not easy to tolerate by the patients and accompanies side effects. Leishmanization showed to be an effective tool against CL and yet is not practised due to drawbacks [35-38]. Use of an appropriate adjuvant accelerates the development of effective prophylactic and therapeutic vaccine against leishmaniasis .
In this study, the gene and protein expressions of TLR2 and TLR4 in macrophages derived from peripheral blood mononuclear cell cultures of patients with healing and non-healing form of lesion were assessed and compared before and after treatment with live Leishmania major to explore a possible role of TLR2 and TLR4 in prolongation of CL lesions induced by L. major.
Materials and methods
The protocol was approved by the Ethical Committee on Human Research, Isfahan University of Medical Sciences. The potential volunteer candidates were informed about the study objectives and the procedure, a volunteer who was willing to participate, sign an informed consent and donate blood sample was recruited.
Two groups of CL patients were selected from the patients referred to Skin Disease and CL Research Center, Isfahan University of Medical Sciences.
Twelve CL patients with healing form of lesion, onset <6 months with no history of treatment for CL, and 12 CL patients with non-healing form of lesion, duration of the lesion(s) more than 1 year and history of at least two full courses of Glucantime injections, were included. Diagnosis was based on the observation of Leishmania using Giemsa-stained smear and/or growth of promastigotes in NNN culture media. Identification of Leishmania causative agent of the lesion was carried out using PCR method as previously described .
Isolation of mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll–Hypaque density gradient (Lymphodex, Fresenius Diagnostics, Wiesbaden, Germany) as previously described . Briefly, PBMCs were washed, and the pellet was resuspended in RPMI 1640 (Gibco, Invitrogen, Karlsruhe, Germany) supp-lemented with 2 mm l-glutamine, penicillin (100 μg/ml), gentamicin (100 μg/ml) and 10% heat-inactivated foetal bovine serum (Gibco), the cell number and viability were checked. PBMCs were divided into two tissue culture plate and concentration adjusted to 5 × 106 cell/ml in each plate. Non-adherent cells were removed after 2 h by washing with PBS. Adherent cells were incubated at 37 °C with 5% CO2 for additional 6 days to allow the cells to differentiate into macrophages . The size of the cells was morphologically increased, and numerous short pseudopods were formed. The cellular phenotype was analysed using flow cytometry and anti-CD14 monoclonal antibody (Abcam, Cambridge, UK) (data not shown), and the purity of the macrophages was about 90 ± 5% of cells express CD14 marker.
Leishmania major (MRHO/IR/75/ER), the same strain that was used for leishmanization and preparation of experimental Leishmania vaccine and leishmanin, was used in this experiment . Parasites were cultures in biphasic NNN medium and subpassaged in RPMI 1640 supplemented with 10% FBS.
Macrophage and parasite interaction
After 6 days, one of the plates of the monocyte-derived macrophages was infected with L. major promastigotes harvested at stationary phase at a ratio of 5:1 parasite/macrophage, and then after 4 h, non-internalized free promastigotes were washed away. The infected cells were incubated in complete RPMI 1640, and then after an additional 12 h of incubation, the adherent cells were scraped using cell scraper.
The other part of the cells was used immediately to analyse TLR2 and TLR4 using flow cytometry, and extra cells were frozen at −70 °C in the presence of RNA protect cell (Qiagen, Hilden, Germany) until used for RNA extraction.
Flow cytometry for expression of TLR2 and TLR4
Treated and untreated cells were washed and resuspended in PBS to quantify cell surface TLR expression, and 5 × 104 cells were used for each sample. Cell surface staining was performed using the following monoclonal antibodies: FITC-conjugated TLR2 (ab13553; Abcam) and FITC-conjugated TLR4 (ab45126; Abcam) antibodies and monoclonal antibody (IgG) FITC (X0932; Dako, Carpinteria, CA, USA) as an isotype control according to the manufacturer's instructions. Briefly, 100 μl of the cells was incubated with 2 μl of anti-TLR antibodies and isotype control for 30 min at 4 °C. Then, the cells were washed twice and resuspended in 500 μl PBS, containing 0.5% formaldehyde. Samples were analysed using BD FACSCalibur flow cytometer using bd cell quest software (BD, Becton-Dickinson, San Jose, CA, USA) (Figs. 1-3).
RNA extraction and cDNA synthesis
Total RNA was isolated using RNeasy Plus Mini Kit (Qiagen) under strict RNase-free condition according to the manufacturer's protocol, isolation included pipetting the cell lysate onto a gDNA Eliminator spin column with subsequent centrifugation. The quality and quantity of RNA were checked using agarose gel electrophoresis and spectrophotometric analysis. cDNA was prepared using a RevertAid First Standard synthesis kit (Fermentas, Vilnius, Lithuania) according to the manufacturer's instruction. The samples were then reverse transcribed using 0.5 μg of RNA under conditions of 65 °C for 5 min, 25 °C for 5 min, 42 °C for 60 min and 70 °C for 5 min.
PCR amplifications were performed using the QuantiFast SYBR Green PCR Kit (Qiagen) in a total volume of 20 μl, containing 1.5 μl cDNA sample, 5 pico moles of each primer and 10 μl of 2x QuantiFast SYBR Green PCR Master Mix. The PCR primers that were used for real-time PCR are shown in Table 1. Real-time reverse transcription PCR (RT-PCR) was run on the ABI Step one Plus (Applied Biosystems, ABI, Foster City, CA, USA). The following cycling conditions were used: initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Melting curve analysis was performed to identify all amplified PCR products. Polymerase chain reactions were performed in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. Relative gene expression normalized to GAPDH, and calculation was performed using method.
Table 1. Human TLR2, TLR4 and GAPDH primers used in real-time polymerase chain reaction
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TLR, toll like receptors.
Statistical analysis was performed using spss version 16. Students paired and independent t-test were used to determine the significant difference; P < 0.001 was considered as significant.
The basic information of the 24 recruited CL patients (M = 21, F = 3) is summarized in Table 2.
Table 2. Basic information and characterization of recruited patients
Age (Mean ± SD)
34 ± 10.7
38 ± 12
Duration of lesion (month)
2.1 ± 1.0
17.3 ± 4.7
No. of the lesion
3.1 ± 1.4
2.5 ± 1.08
Flow cytometry of TLR2 and TLR4 on macrophages surface
The expression of TLR2 on the surface of macrophages in healing and non-healing form of CL was quantified using flow cytometry analysis. Cell surface expression of TLR2 in healing and non-healing form of CL before stimulation was 58.00 ± 15.00 and 32.74 ± 7.72, respectively, which was significantly different (P <0.001). The level of TLR2 on the surface of macrophages after stimulation in healing and non-healing form of CL was 82.50 ± 12.33 versus 49.70 ± 11.36, which was significantly different (P <0.001). Mean fluorescence intensity (MFI) for TLR2 before and after stimulation in healing and non-healing form of CL was also significantly different (P < 0.0001). The differences between TLR2 surface expression before and after stimulation in patients with healing form of CL were 24.50 ± 5.06 versus 16.96 ± 4.92 in non-healing form of CL. The data were shown that the difference of TLR2 in healing form of CL was significantly higher than non-healing form of CL (P = 0.0034) (Fig. 4). TLR4 was also analysed using flow cytometry for cell surface expression. Cell surface expression of TLR4 in healing and non-healing form of CL before stimulation was 37.40 ± 7.91 and 29.64 ± 16.06, respectively, which was not significantly different (P = 0.1876) between the two groups. The level of TLR4 on the surface of macrophages after stimulation in healing form of CL was 58.30 ± 6.46 versus 41.80 ± 15.35 in non-healing form of CL, which was significantly different (P = 0.0064). The mean fluorescence intensity (MFI) of TLR4 before and after stimulation in healing and non-healing form of CL was also significantly different (P < 0.0001). The differences between TLR4 surface expression before and after stimulation in patients with healing and non-healing form of CL were 20.63 ± 4.84 and 12.16 ± 3.33, respectively. The data were shown that TLR4 in patients with healing form of lesion was significantly higher than TLR4 in patients with non-healing form of lesion (P = 0.002) (Fig. 4).
Real-time PCR TLR2, TLR4 gene expression
mRNA levels of TLR2, TLR4 and GAPDH as housekeeping gene were quantified by quantitative real-time PCR. The mean expressions of TLR2 mRNA were 6.40 ± 1.05 and 3.70 ± 1.16 in patients with healing and non-healing form of lesion, respectively, which displayed a significant higher in healing form of CL (P < 0.0001) (Fig. 5). The mean TLR4 mRNA expression was 3.44 ± 1.13 and 2.24 ± 0.98 in patients with healing and non-healing form of lesion, respectively, which was significantly different (P = 0.0210) (Fig. 5).
Despite of numerous publications still process of healing and protection in human leishmaniasis, exact mechanism of APCs and the development of protective immune response against the disease are largely unknown. TLR activation plays an important role in induction of macrophages cytokines and adaptive immune response, which promote Th1 cell differentiation [30, 41].
Although the importance of TLR activation in macrophages in interaction with Leishmania infections is being widely studied in animal model [30, 32-34, 41, 42], the information of the role of TLRs in disease progress, lesion healing process and protection in human leishmaniasis is not well established.
In the current study, the level of TLR2 and TLR4 expression was assessed to explore possible relationship between TLR2 and TLR4 expression and lesion healing in patients with CL caused by L. major. The results showed that the cell surface expression of TLR2 and TLR4 in both groups of patient was elevated after coculture of macrophages with L. major, and TLR2 surface expression on macrophages before stimulation was significantly higher in patients with healing form of lesion in comparison with patients with non-healing form of lesion, but TLR4 surface expression was not differ between the two groups before stimulation. A possible explanation is that in initial phase of infection, TLR2 may be more important in recognition of Leishmania than TLR4. Moreover, the difference (delta) of cell surface expression of TLR2 and TLR4 before and after stimulation was significantly different (P < 0.001) between the macrophages of both groups of the patients. Real-time PCR used to measure the levels of TLR2 and TLR4 mRNA, and the results showed that the mean relative gene expression of TLR2 and TLR4 in patients with healing form of lesion was significantly higher than the patients with non-healing form of lesion (P <0.001), (P = 0.0210). The reason for the difference might be due to different interaction between the TLR molecules and Leishmania surface Ags such as LPG. Another possibility is that different intracellular signalling pathways or adaptor molecules might be also involved . This alteration may result from a difference in regulatory factors, enhancers and inhibitor molecules, which trigger by the innate and adaptive immune responses [19, 25, 43].
It was shown that BALB/c mice infected with L. chagasi induce TLR2 and TLR4 mRNA expressions throughout the course of infection, and a higher expression of TLR4 and TLR2 was recorded at the peak of parasitemia. It was suggested that L. chagasi interacts with TLR2/TLR4, and the infection induces cytokine modulation during the acute and chronic phases of the disease . In another study, it was shown that L. pifanoi amastigotes induced lower levels of cytokines in macrophages in the absence of TLR4 and higher IL-10/IFN-γ ratios in TLR4-deficient mice than in BALB/c mice . Studies using TLR4−/− mice indicated a protective role in L. major infections , and this group of mice was clearly less efficient to control parasite at the site of infection when compared with wild-type mice . More recently, it was reported that TLR2−/− mice are also less susceptible to the infection with L. amazonensis than the wild-type C57BL6, and also a lower parasite burden and less inflammatory cells were shown in the first weeks of infection , which suggest that depend upon Leishmania species TLR2 might play a role in disease establishment.
It was shown that purified L. major lipophosphoglycan (LPG) upregulated both mRNA and the membrane expression of TLR2 in NK cells , and LPGs of L. major, L. mexicana and L. aethiopica were identified as TLR2 ligands in animal model .
It was also reported that LPG stimulates cytokine production by human monocytes via TLR2 . Later was reported that the induction of NO production by macrophage cell lines challenged with LPG was dependent upon TLR2, but not TLR4 signalling . There are some evidence that a part of LPG structure induced IL-12 and Th1 response in animal model through TLR2, showing that TLR2 was stimulated by Leishmania LPG and developed the inflammatory responses in mice . In the case of visceral leishmaniasis, L. donovani amastigotes antigens upregulated TLR2 expression and induced the activation of TLR-signalling proteins in macrophages .
Recently, a study of TLR4 mutations in patients with CL showed that genotypes of TLR4 molecule are important in individuals with chronic or acute disease when compared with non-infected individuals, suggesting that TLR4 polymorphism might be related to susceptibility or severity of the disease .
Recently, it was reported that TLR2 plays a role in initiation of proinflammatory response during visceral leishmaniasis, pretreatment with arabinosylated lipoarabinomannan (Ara-LAM) induced TLR2 expression in L. donovani-infected macrophages, activation downstream signalling via (MyD88) mediated and induction of the proinflammatory response .
It was also shown that miltefosine treatment led to a significant induction of TLR4 and TLR9 in L. donovani-infected THP1 cells in comparison with untreated infected cells, and miltefosine treatment induced a proinflammatory cytokine response in infected THP1 cell line .
It also reported that activation of TLR4 during immunotherapy for leishmaniasis has been related to the development of cure rate in animal models , indicating the relationship between TLR and immunotherapy for Leishmania infection.
In particular, these findings show a protective role for TLR2 and TLR4 during Leishmania infection and an effective Th1 response.
The outcome of Leishmania infection in CL patients for completely unknown reasons varies from spontaneous self-healing lesion to non-healing lesion refractory to all types of available treatments, and the current study clearly shows a significant different in TLR2 and TLR4 in protein and gene expression level, which might implicate that TLR molecules may play a role in healing of the lesion or progression of the lesion. Further studies with a larger sample size are required to explore the role of innate immune response in CL lesion. This information is helpful for immunotherapy and vaccine development.
The study was supported by Isfahan University of Medical Sciences (Grant No. 188051). The authors would like to appreciate Dr Gilda Eslami and Miss Maryam Peymani for sincere technical assistance and also Miss Leila Shirani and other staffs of Research Center for Skin Disease and Leishmaniasis (Sedigheh-Tahereh) for kindly collaborations. The authors declare no conflict of interests.