Therapeutic and Preventive Effects of Morphine Against Leishmania Major and Evaluation the Expression of TLRs and Cytokines in Infected Macrophages in Vitro and in BALB/c Mice

Purpose: Toll-Like Receptors (TLRs) and cytokines play key roles in infection control. They also enhance phagocytosis and killing of parasites. Morphine can modify host immunity and defense against infectious diseases. Methods: In this study, we assessed Therapeutic and preventive effects of morphine against Leishmania major and then we evaluated the expression of TLRs and pro and anti-inamatory cytokines in both healthy macrophages and those infected with Leishmania major in vitro and in BALB/c mice. Results: Morphine showed preventive effect and no lesions were observed in the group that was taken morphine before being challenged with promastigotes. TLR2 expression decreased in drug-treated healthy macrophages, whereas TLR4 expression increased. TLR7 expression decreased in healthy macrophages. TLR9 expression was the highest in the groups treated with morphine in infected macrophages. Our ndings revealed that healthy macrophages produce higher TNFα and lower IL6; the infected macrophages show a reverse pattern by producing higher IL6 and lower TNFα. We found that treatment with morphine strengthen defensive reactions against leishmaniasis. In mouse macrophages, the highest level of TLR9 expression was induced by morphine. Conclusion: TLR9 has critical role for recognition and control of microbial infection. No lesions were developed in mice treated with morphine before challenge which suggests a protective role for morphine in leishmaniasis. The positive role of morphine in decreasing IL-10 expression and increasing the expression of inammatory cytokines such as TNF-α and therefore its preventing role in Leishmania disease.


Introduction
Leishmaniasis is a vector-borne disease caused by various species of Leishmania, has several clinical manifestations and is one of the most prevalent health problems in endemic countries (1). Treatments for leishmaniasis can have signi cant side effects and in some cases, drug-resistance has become a problem. Therefore, new drugs with high e cacy and fewer side effects are needed.
Following invasion, promastigotes undergo phagocytosis by macrophages, but survive to differentiate into inactive amastigotes which concentrate inside phagosomes of macrophages to multiply (2). Opioids have been shown to have immuno-modulatory effects on various infections. Morphine can kill the parasite by reactivation of nitrogen intermediates or induction of iNOS to produce NO as an important defensive mechanism against Leishmania spp (3)(4).
Infection triggers defense mechanisms by pattern recognition receptors (PRRs) to identify pathogenassociated molecular patterns (PAMPs) of pathogens. The family of Toll like receptors (TLRs) is a critical group of PRRs associated with both innate and adaptive immune systems (5). TLRs activate macrophages and play a key role in infection control (6). They also in uence the innate and adaptive immunity by enhancing phagocytosis and killing of parasites (7).
TLRs are intermembrane receptors that identify pathogens based on speci c molecular patterns and have a potential role in inducing an innate immune response by activating different pathways (8). Modulation of host TLR expression and activation protects against exacerbation of the disease (9).
The relation between in ammation and immunity responses is complex; however, it is of great importance in disease detection. Like TNFα, IL1 is a basic pro-in ammatory cytokine, which acts as a warning cytokine secreted by macrophages (10)(11). IL10 has multifunctional activities that affect immunity regulation and in ammation. This cytokine reduces the expression of Th1 cytokine, MHC antigens and macrophages' stimulant assistance molecules (12). IL12 is made by two sub-units of 35 and 45 KDa, and plays an essential role in resistance against L. major (13). Morphine and opioid combinations affect the patient's behavior and mood by relieving pain. They also balance various immunity system activities. It has been shown that endogen opioid peptides such as endorphinβ-often suppress the prevention effects. However, different concentrations of morphine have different effects on the immunity systems (14)(15). Experiments revealed that long term intake of morphine weakens the normal functions of the immunity system, especially in macrophages and lymphocytes (16-17).
Morphine in low dose have protective effect and can increase immune responses against infection. In our earlier in-vitro study, we showed that treatment with morphine at low doses inhibits the growth of amastigotes inside macrophages (18).
Therefore, in the current in vivo work, we have used low doses of morphine for prevention and treatment of leishmaniasis caused by L. major in BALB/c mice. Additionally, the effects of morphine and glucantime, a standard antimony-containing anti-leishmanial compound have been investigated for expression of TLRs (TLR1, TLR2, TLR3, TLR4, TLR7, TLR9). In previous study we found that morphine has preventive and therapeutic effects on toxoplasmosis (19). Also, we investigated the effect of morphine, on the expression of cytokines IL1, IL6, IL10, TNFα, IL12 p35and IL12 p40 by macrophages infected with amastigotes of L. major. Moreover, the same effects were studied on healthy macrophages in vitro, as well as in animal model of BALB/c. Drug assay 12-well plates containing RPMI 1640 enriched with FBS (10%), penicillin (100IU/ml), and streptomycin (100µg/ml) were used to culture J774 macrophages (1×10 5 cells/well) and incubated at 37°C in 5% CO2 atmosphere. In this part of the study, we used two separate experiments. In the rst experiment the macrophages were exposed directly to various concentrations of drugs according to Table 1. In a second experiment the macrophages were rst infected with promastigotes of L. major (1×10 6 per well) in stationary phase (1:10 ratio) and incubated for 24 h. After infection, the supernatant was discarded and the different concentrations of drugs listed in Table 1 were added. The plates were again incubated under the same conditions for a further 24 h. The macrophages were collected from each well by centrifugation at 1500 rpm for 10 min. The macrophage pellets were washed with cold PBS and stored for later RNA extraction and cDNA preparation. The cDNA was used for assessment of TLR expression by real time PCR (20)(21). Female BALB/c mice (120 mice, 6-8 weeks old) were purchased from Pasteur Institute of Iran. The mice were kept at a controlled temperature (22 ± 1˚C) and humidity (50 ± 10%) with a light/dark cycle of 12/12 h. The protocol of study was approved by Medical Sciences Ethical Committee of Tarbiat Modares University with number 52/D/8207. Food (pelleted concentrate) and water were provided ad lib. Infection was carried out by injecting 2×10 6 stationary phase promastigotes subcutaneously in the tail base of mice. Ten mice were allocated to each treatment group (12 groups) as follows:

Materials And Methods
1. Infected with L. major as a control group (no treatment) 2. Uninfected control group (no treatment) 3. Infected group given a subcutaneous injection of morphine (1 mg/kg) weekly for four weeks before being infection with L. major promastigotes (to evaluate the protective effect of morphine). 4. Morphine treated group challenged with subcutaneous injection of morphine (1 mg/kg) weekly for four weeks. 5. Uninfected group challenged with subcutaneous injection of morphine (1 mg/kg) weekly for 4 weeks.
Five mice of each group were sacri ced 4 weeks after completion of treatment (8 weeks after challenge) and the spleens were removed for evaluation the spleen parasite burden, lymphocyte cytokine assay and expression of TLRs by peritoneal macrophages. The remaining 5 mice in each group were followed for up to 22 weeks for measuring changes in lesion size and survival rate.
Spleens were immersed in a 6 cm-plate containing 5 ml cold sterile PBS solution (+ 4 0 C). Then, one third of each spleen was separated and transferred into screw cap tubes for parasite load determination and real time PCR, while the remainder was used for cytokine assay.

Cytokine assay
To investigate the stimulation of IL-4 and IFN-Y cytokines, spleen lymphocytes were extracted and cultured with SLA (soluble Leishmania antigen). Brie y, splenocytes were separated under sterile conditions and cultured in 24-well plates (3.5×10 6 cell/ml) with 25 µg/mL of SLA. Plates were incubated at 37°C under 50% CO 2 for 72 hours. The supernatant was collected and 300 µl was removed and kept at -70°C until used (20)(21).
U-CyTech kit (Netherlands) was used to investigate the expression of IL-4 and IFN-γ in the lymphocyte cell supernatants of infected and control groups. The stock materials and solutions were prepared according to the manufacturer's instructions. The kit standard was applied to produce a standard curve, based on which the test samples were evaluated.
Parasite burden assay by quantitative Real Time PCR Fresh spleen tissue (25 mg) was placed in a 1.5 mm tube and homogenized with a mortar and pestle. DNA extraction was done using Cinna Pure One kit (Sinagen Co) according to manufacturer's instructions. DNA concentrations were determined in ng/µl. the purity of DNA was ensured by measuring A260/A280 ratios. Parasite load was evaluated by quantitative Real Time PCR. Six standards from 10 to 10 6 copy number of parasites were prepared and according the Cycle of Threshold (CT) a standard curve was drawn. The parasite load was evaluated for all treated and control groups using Genesig® Leishmania Advanced Kit.

Lesion size and survival rate measurement
During treatment and up to 22 weeks after treatment, diameter of lesions was measured weekly using Vernier calipers. Survival rates for the remaining ve mice from each group were recorded weekly followed for 22 weeks.
Extraction of macrophages for expression of TLRs assessment J774A.1 macrophages and peritoneal macrophages were used for TLRs expression of TLRs assessment and extracted RNA was used to build cDNA. Real time PCR was also performed for in vitro assays.

Preparation of cDNA and real time PCR
Macrophage pellets were used to extract RNA using the Qiagen kit (RNeasy Mini Kit, Qiagen, Germany) and the amount of extracted RNA was measured by a Biophotometer device. Subsequently, extracted mRNA was used to construct cDNA using QuantiTect Reverse Transcription kit, (Qiagen Germany). The cDNA (10 ng) was ampli ed using speci c TLRs primers as well as Real Q Plus 2x Master Mix Green Qiagen Mastercycler was used to perform the quantitative real-time PCR assays. The relative fold change of gene expression was analyzed by comparative cycle threshold (2 − ΔΔCt ) method. The data of target gene expression were normalized against the expression of reference genes including GAPDH and β 2 microglobulin. Changes in test group relative to those in control group was assessed based on changes in target gene expression (22).
Chao CC, Gekker G, Sheng WS, Hu S, Tsang M, Peterson PK. (1994) Priming effect of morphine on the production of tumor necrosis factor-a by microglia: implications in respiratory burst activity and human immunode ciency virus-1 expression. J Pharmacol Exp Ther 269:198-203.

Real-Time PCR of cytokines
In this study, Qiagen Master-Cycler was used to evaluate quality of Real-Time PCR. GAPDH and β 2 microglobulin were applied as housekeeping genes. The RealQ Plus 2X Master Mix Green, without ROX (Ampliqoun, Denmark) was used to analyze cytokines (IL1, IL6, IL10, TNFα, IL12 p35and IL12 p40). The test was performed as per following conditions: 95 0 C for 15 min (95 0 c for 15s, 60 0 c for 30s, 72 0 c for 30s) for 40 cycles. Expressions of IL1, IL6, IL10, TNFα, IL12p35, and IL12p40 were normalized using B2 microglobulin and GAPDH, and measured using fold changes with 2 − ΔΔCt method. The sequences of the relative primers were as follows. Four weeks after parasite challenge t, the size of lesions were recorded weekly and statistically analyzed. In addition, humoral and cellular immune-adsorbent assays were also applied and statistically analyzed. Kolmogrov-Spirnov test was used to ensure data normalization and the One-Way ANOVA and LSD were applied to compare the data. Mortality and survival rates between the groups of mice were assessed after challenge. The con dence interval was set to 95% and p ≤ 0.05 was considered statistically signi cant. SPSS software was used for statistical analysis.
All experiments were conducted in triplicate wells. The data are presented as mean ± SD. The data were analyzed using GraphPad Prism, version 5.0 and online site RT 2 -Pro ler at (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). Student's t-test was performed to check the difference between means of the representative treatments. Differences were considered signi cant at P-value < 0.05.

Parasite burden
The parasite load for all groups was measured by comparing with the standard curve and the results are shown in Table 2. The greatest reduction was seen in group that received morphine before being challenged with parasites. The results of cytokine assays and IFNγ/IL4 ratios are presented in Table 3 and Fig. 1. In the group that was treated with morphine before promastigote challenge the IFNγ/IL4 ratio was (2.51) and is approximately same to healthy control group (2.62). Lesion size and survival rate The lesion sizes for all groups are shown in Fig. 2. No lesions were observed in the group treated with morphine before being challenged with promastigotes. Table 4 summarizes the reduction in lesion diameters at 7 and 12 weeks post-challenge, compared with those in the control group. The survival rate increased in all treated groups in comparison to control. The highest survival rate was in those treated with morphine before challenge (Fig. 3).

Expression of TLRs in macrophages
Intact macrophages (β 2 microglobulin normalized) A signi cant decrease in expression of TLR1 and TLR2 was observed in all experimental groups. In case of TLR3, a signi cant decline in gene expression was detected in all groups except for the 0.1 µg/ml dose morphine treated group (P < 0.05) (Fig. 4).

Intact macrophages (GAPDH normalized)
A marked and statistically signi cant decrease in expression of TLR1, 2 and 4 was observed. In the case of TLR3, a reduced gene expression was noticeable in all groups except in the 0.1 µg/ml morphine treated group. The expression of TLR7 and TLR 9 were the same as with β2 microglobulin normalization (Fig. 4).
Macrophages infected with L. major (β 2 microglobulin normalized) TLR1 and TLR3 expression increased in all treated groups compared to control. A signi cant expression decrease pattern was observed for TLR4 and TLR7 in all treated groups. TLR9 showed signi cant decrease in the morphine treated group (100 µg/ml) (Fig. 5).
Macrophages infected with L. major (GAPDH normalized) TLR1 and TLR3 expression was signi cantly enhanced in all treated groups. TLR2 expression declined in all except the 10 µg/ml morphine and 100 µg/ml morphine treated groups. TLR4 and TLR7 expression was signi cantly decreased in all groups (Fig. 5).

Mouse macrophages (β 2 microglobulin normalized)
The expression of TLR1 decreased in healthy mice, morphine-treated healthy mice and morphine-treated infected mice. The receptor expression showed an increasing pattern in all other groups. TLR2 expression signi cantly increased in all groups. However, TLR3 demonstrated a decreasing expression pattern only in morphine-treated infected mice. TLR4 expression increased in all mice groups. The expression trend of TLR7 was increasing in all groups other than the morphine-treated mice. TLR9 showed higher expression in all groups except in the morphine-treated infected mice as well as in healthy mice (Fig. 8).

Mouse macrophages (GAPDH normalized)
All the groups showed a signi cant increase of TLR2 expression. Healthy mice as well as infected mice treated with morphine showed a decline in expression pattern for TLR4. In infected mice groups treated with morphine, TLR7 expression declined, while other groups showed increased expression. In healthy mice and infected mice treated with morphine TLR9 expression was low compared to other groups ( Fig. 6).

Cytokines expression in uninfected macrophages
The groups were normalized with either internal control genes GAPDH or β 2 microglobulin. Amongst groups normalized either with GAPDH or β 2 microglobulin, IL1 expression showed reduced in all groups.
Also, signi cantly higher levels of IL10 expression were observed in groups treated with glucantime, with either GAPDH or β 2 microglobulin than the control group (P < 0.05). Compared with the control group, meaningful increases in the levels of IL12 P35 expression were noted in groups treated with morphine 0.1 and glucantime, from among those normalized with either GAPDH or β 2 microglobulin. In other groups, signi cant reduction of IL12 P35 expressions were recorded. Concerning IL12 P40, both normalized groups with the genes and treated with morphine 0.1 showed increase of expression with a signi cant difference (P < 0.05) (Fig. 7).
Cytokines expression in macrophages infected with amastigote IL1 expression increased in both groups treated with morphine 10 and glucantime, but the level of expression was signi cantly different only in the latter group compared with genes β 2 microglobulin (P = 0.0384) and GAPDH (P = 0.0386). In this respect, no signi cant difference was observed between control group and morphine 10 treated group. IL6 expressed at signi cantly higher levels in groups treated with morphine (10 and 0.1) than in the control group (P < 0.05). Group treated with morphine 10 showed signi cant increases of IL10 expression relative to the control group. Other groups showed decreased expression in comparison with the control group (P < 0.05). As far as TNFα expression is concerned, groups treated with morphine 10 and glucantime alone recorded signi cantly higher titers than the control group (P < 0.05). On the other hand, the group treated with morphine 10 alone produced signi cantly less TNFα than the control group. All other groups normalized either with β 2 microglobulin or GAPDH indicated signi cantly reduced expression compared with the control group. However, IL12 P40 expression was signi cantly lower in groups normalized either with β 2 microglobulin or GAPDH than the control group (Fig. 8).

Cytokine expressions in macrophages of BALB/c mice
The contrary was observed in other groups. Moreover, except of UM with insigni cant difference relative to the control group, other groups showed meaningful elevated level of IL6 expression.
In the group under treatment with morphine and glucantime, a meaningful reduction of expression was seen in comparison to that in the control group, (P < 0.05). The morphine-treated group showed reduction of IL12 P 35 expression in comparison with the groups normalized with β 2 microglobulin and GAPDH (P = 0.0242 and P = 0.002 respectively).
There was signi cantly elevated level of IL12 P 35 expression in the group treated with glucantime as compared with the control group (Fig. 9).

Discussion
Leishmania parasites are obligate intracellular pathogens. Therefore, macrophages provide necessary media for the parasite survival, reproduction and differentiation. Macrophages attack the invading parasites so that their responses determine the initiation of the infection process (23)(24).
Drug addiction causes some changes in the host immune system. Clinically, patients receiving chronic opioid treatment as well as opioid addicted people have impaired immune systems and are more vulnerable to infections (25)(26). Hence, the result of immunosuppression increases host sensitivity to infections. Experimental studies have shown that long term morphine consumption weakens the normal function of the immune cells, speci cally macrophages and lymphocytes (27)(28).
Morphine has therefore been shown to have both debilitating and stimulating effects on immune system.
In an earlier study, we found that low doses of morphine enhanced immune function and could control amastigote multiplication in infected macrophages whereas high doses increased the growth of Leishmania major amastigotes (18). Morphine can therefore increase endogenous and exogenous immune defense responses. Importantly, opioids do not have cytotoxic effects and do not damage cancer cells directly. However, they inhibit their growth and allow immune mechanisms to destroy them. Opioid Growth Factors (OPG) in combination with chemotherapy can therefore produce positive effects on patients (38).
In our study, the highest parasite load was in the untreated infection control group. Within all the treatment groups, those treated with morphine before leishmanial infection not only had the lowest parasitic load but also did not develop any lesions. This clearly demonstrates the preventive and/or adjuvant role of morphine when used at low doses.
The highest survival rate was also observed in the pretreatment group and was similar to that of healthy control group.
Cellular immunity was evaluated by cytokine measurement. We found that the response of host immune system determines the progression or improvement of leishmaniasis. Where the immune system responds through cellular immunity, this may lead to improvement and inhibition of the disease. Hence, calculation of the ratio of interferon gamma (indication of cellular immunity response) to interleukin-4 (indication of humoral immunity response) and the comparison of change after parasitic challenge in both treated and control groups can help in evaluating immune response processes.
It seems that TLR2 is more intensely activated against metacyclic promastigotes of Leishmaniasis major than against non-infectious procyclic parasites. LPG on the surface of promastigotes plays a key role as an effector in macrophage attachment and penetration, NK cells and TLR2 activation (39)(40), modi cation of nitric oxide production (NO) (41), inhibition of protein kinase C (42), activation of extracellular neutrophil (43), as well as parasite attachment to midgut of vectors (44). It has been suggested that TLR2 signal transmission induces PKR activation (45), and that leishmania LPG is a TLR2 ligand. Apparently, therefore, TLR2 signaling is of great importance in leishmaniasis. It has been shown that TLR2 knockout mice infected with Leishmania braziliensis develop smaller lesions and produce lower levels of IL-10 (46  (50). Another study suggested that TLR4 detects combination of proteoglycolipid P8 in Leishmania pifanoi and plays a key role in infection control (51). However, there is no evidence as yet of a direct reaction between the molecules derived from Leishmania major and TLR4. However, TLR4 was shown to be essential for immunity responses against Leishmania major parasites by both innate immunity and acquired immunity (48).
Granuloma formation is an important healing process in cutaneous leishmaniasis. Histological research indicated that the proper adaptive immune response is compatible with IFNγ. However, parasitic drug resistance in dermatitis, particularly in case of recurrence of leishmania ulcers is due to immunosuppression (52). Reimmunization was used to examine TLR9 expression by giant cells of epithelioid cells characteristics, although dendritic cells may do the same. TLR9 is important for formation of tuberculosis granuloma, liver and lung granulomas (53). Also, those group treated with 0.1 µg/ml morphine demonstrated the highest gene expression. Groups treated with glucantime and the group get morphine before infection showed elevated expression. Signals of TLR2 change Th2 cellmediated dermatitis to chronic in ammation by induction of IL-4 and suppression of IL-10 (54).
Activating TLR2 in skin results in interleukin-6 (IL-6) production that triggers the suppression of MDSC (myeloid-derived suppressor cells) and then suppress the responses of T cells (55).
In addition, we can consider the use of morphine at low concentrations as an adjuvant in vaccination against leishmaniasis. Our previous study showed that when uninfected macrophages were exposed to different concentrations of morphine (0.1, 10 and 100 µg / ml), cytotoxicity was low with 91.19%, 95.71% and 92.64% viability after 24 hours respectively (56).
Previously, the expression of TLR2 and TLR4 from extracted RNA lesions of groups treated with morphine was studied. This showed increased TLR2 gene expression in all treated groups compared to control (57).
TNFα is a cell signaling protein involved in systemic in ammation. The cytokine is produced by activated macrophages to provide acute phase reactions. IL12 is a basic cytokine that differentiates Th1 cells in Leishmania. It is mainly produced by macrophages, monocytes, dendritic cells and B cells (58 Mean of lesion diameters (mm) after treatment (4 weeks after challenge) until 17 weeks after challenge of all groups treated and control groups.