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Leptin augments protective immune responses in murine macrophages and enhances potential of miltefosine against experimental visceral leishmaniasis
Accepted Manuscript Title: Leptin Augments Protective Immune Responses in Murine Macrophages and Enhances Potential of Miltefosine against Experimental Visceral Leishmaniasis Author: Rahul Shivahare Wahid Ali Preeti Vishwakarma S.M. Natu Sunil K. Puri Suman Gupta PII: DOI: Reference:
S0001-706X(15)30045-0 http://dx.doi.org/doi:10.1016/j.actatropica.2015.06.024 ACTROP 3666
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
5-3-2015 19-6-2015 22-6-2015
Please cite this article as: Shivahare, Rahul, Ali, Wahid, Vishwakarma, Preeti, Natu, S.M., Puri, Sunil K., Gupta, Suman, Leptin Augments Protective Immune Responses in Murine Macrophages and Enhances Potential of Miltefosine against Experimental Visceral Leishmaniasis.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2015.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Leptin Augments Protective Immune Responses in Murine Macrophages and Enhances Potential of Miltefosine against Experimental Visceral Leishmaniasis Rahul Shivaharea*, Wahid Alia, Preeti Vishwakarmab, S.M. Natua, Sunil K. Purib, Suman Guptab a
b
Department of Pathology, King George’s Medical University, Lucknow- 226 003 (India)
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow- 226 031 (India)
*Corresponding author Department of Pathology, King George’s Medical University, Lucknow- 226 003 (India) E-mail addresses: [email protected]; [email protected] Phone: +91 522 2257580, +91 9935300350; Fax: +91 522 2257580
Highlights
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Leptin reduces effective concentration of miltefosine by two-folds.
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Leptin induces host macrophages for the activation of Th1 biased immune responses.
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Leptin when combining with miltefosine boosted up host immunity.
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Leptin plus miltefosine heightened the phagocytic activity of macrophages.
Abstract Adverse side effects and drug resistance issues are the two most important drawbacks which influence the widespread use of existing antileishmanial drugs. Use of immune stimulating agent with standard antileishmanial might be helpful to minimize the toxic effect of drug, shorten the dose regimen and delay the emergence of resistance. In the present study, we explored the in vitro immunomodulatory potential of an immunomodulator, leptin with lower concentration of standard drug, miltefosine. The level of Th1/Th2 cytokines, production of nitric oxide and reactive oxygen species and phagocytic activity were assessed by ELISA, Griess reaction and flow cytometric analysis, respectively. Leptin at a concentration of 15 µg/mL showed heightened level of Th1 cytokines and nitric oxide generation from murine macrophages (J-774A.1 cells). Leptin (15 µg/mL) also reduces the effective concentration of miltefosine by 2-folds from 7.5 µM to 3.7 µM. When given in conjunction with lower concentration of miltefosine (4 µM), leptin (15 µg/mL) was significantly (***p<0.001) elevated the level of IL-12 (7.7 fold), TNF-α (8.1 fold) and nitric oxide (6.6 fold) along with markedly (***p<0.001) suppressed level of IL-10 and TGF-β when compared with untreated infected macrophages. Leptin plus miltefosine also induces the phagocytic ability (**p<0.01) of macrophages in comparison to leptin alone and miltefosine alone treated groups. These finding illustrate that leptin activates host macrophages to generate protective immune response for the successful elimination of Leishmania parasite at lower concentration of miltefosine and has potential for further exploration in experimental animal model of visceral leishmaniasis (VL). Keywords: Leishmania donovani; J-774.A1 cells; Leptin; Miltefosine; Combination therapy; Immune response
1. Introduction Visceral leishmaniasis (VL) or black fever (kala-azar in Hindi) is a neglected infectious disease caused by the protozoan parasite of the genus Leishmania in tropical and subtropical regions particularly in the Indian subcontinent, East Africa and South America (Guerin et al., 2002). VL threatens 200 million people in 62 countries with an estimated 5,00,000 new cases and 60,000 deaths each year (Desjeux, 2004). This systemic infection affects the poorest people mainly in rural areas and it is a main public health problem in Indian sub-continent with more than 60% of global cases (Alvar et al., 2012). VL is potentially fatal disease in which parasite manipulates the normal immune function of the host for the survival and reside within the macrophages of spleen, liver and bone marrow (Stanley and Engwerda, 2007; Murray et al., 2003). Leishmania infected host macrophages are incapable to produce inflammatory responses which involves production of tumor necrosis factor (TNF)-α, interleukin (IL)-12- dependent production of interferon (IFN)-γ and subsequent generation of nitric oxide (NO) (Olivier et al., 2005). In addition, Leishmania parasite can stimulate the macrophages for the production of immunosuppressive molecules, IL-10, transforming growth factor (TGF)-β and prostaglandin E2 (PGE2). These molecules directly or indirectly distort normal immune function and favor parasite survival within the host macrophages (Olivier et al., 2005). The co-infection of HIV is exacerbated the morbidity and mortality rate in immuno-compromised VL patients (Tiuman et al., 2011). Currently, antileishmanial chemotherapy relies on handful number of drugs including pentavalent antimonials, amphotericin B and its formulations, paromomycin and oral drug miltefosine. These all therapeutic options are unaffordable to poor patients, have adverse side effects and drug resistance issues (Alvar et al., 2006; Croft et al., 2006). None of the medicaments are effective in all cases of VL infection. In recent years, several reports indicated that combinations of existing antileishmanial drug with another antileishmanial agent or with
immunomodulators are advantageous over monotherapy as they minimize the toxic effect of drug, shorten the dose regimen and delay or prevent the emergence of resistance (Seifert and Croft, 2006; Musa et al., 2012; Omollo et al., 2011; Olliaro, 2010). Since, diminish immune function is responsible for the progression of Leishmania infection in host, immunomodulator along with chemotherapy might be beneficial to boost up host immune cells to generate protective immune response for the successful treatment of VL at short dose regimen. Some potential immunomodulators such as imiquimod with paromomycin (El-On et al., 2007), tamoxifen with amphotericin B (Trinconi et al., 2014) and CpG-ODNs with miltefosine (Sane et al., 2010; Shivahare et al., 2014a) have been explored for the successful treatment of cutaneous and visceral forms of leishmaniasis. In the present study, we have demonstrated a new combination of a potent immune stimulating agent, leptin and oral drug, miltefosine. Leptin is an adipocyte derived hormone which has pro-inflammatory effects on T cell populations. It is reported that leptin stimulates macrophages and natural killer cells for the production of pro-inflammatory Th1 cytokines such as IL-2, IFN-γ, TNF-α and IL-18, with resultant decreased production of the Th2 cytokines, IL-4, IL-5 and IL-10 (Lord et al., 1998; Martin-Romero et al., 2000). Considering these interesting reports, we explored the effects of combination therapy in parasite survival, alterations in immunological and biochemical parameters viz. Th1/Th2 cytokines, production of NO and reactive oxygen species (ROS) and phagocytic activity in Leishmania infected murine macrophages (J-774A.1 cell line) when incubated/ treated with leptin alone or in combination with lower concentration of miltefosine.
2. Materials and Methods 2.1. Parasite, cell culture and compounds
The WHO reference strain of Leishmania donovani (MHOM/IN/80/Dd8) was maintained in the laboratory as promastigotes in vitro in M-199 medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS) at 24ºC ± 2ºC. Murine macrophage cell line (J-774A.1 cells) obtained from National Centre for Cell Sciences, Pune (India) was cultured in RPMI-1640 medium (Sigma-Aldrich, USA) supplemented with 10% heat inactivated FBS at 37°C in a 5% CO2 incubator. Leptin (Peprotech, USA) and miltefosine (Synphabase AG, Switzerland) were dissolved in deionized water and phosphate buffer saline (PBS) respectively prior to in vitro experiments. 2.2. In vitro assessment in macrophage- amastigote system Mouse macrophages (J-774A.1 cells) (2 x 104/mL) were seeded in complete RPMI medium and layered in 16 well chamber slides (Nunc) under a final volume of 100 µL/well and allowed to adhere overnight at 37°C in a 5% CO2-95% air mixture. Macrophages were infected with stationary phase promastigotes at a macrophage-promastigote ratio of 1:10 on day 2. Infected culture was maintained at 37°C in a 5% CO2 incubator. Promastigotes were phagocytized by the macrophages and inside the phagolysosomes, they were transformed into amastigotes (nonmotile form). On day 3, test compounds were prepared in complete RPMI medium and added at two fold dilution in complete medium at different concentrations after replacing the previous medium and the slides were incubated at 37°C in a 5% CO2 incubator. Compounds containing medium were changed on day 5 and slides were incubated at 37°C in a 5% CO2 incubator. Finally slides were fixed with 100% methanol and stained with 10% Giemsa stain for 45 min on day 6 (Seifert et al., 2006; Gupta et al., 2002). The number of amastigotes per 500 cell nuclei was counted in each well and the parasitic burden is expressed in terms of the number of amastigote per 100 cell nuclei. Drug activity (percent inhibition) was determined by comparing amastigote count of treated and untreated wells by the general formula:
Percent Inhibition (PI) =
Where, N is average number of amastigotes per 100 cell nuclei of untreated well and n is average number of amastigotes per 100 cell nuclei of treated well. 2.3. Estimation of cytokine levels The level of various cytokines secreted by murine macrophages was measured using a BD OptEIA ELISA kit (BD Biosciences, USA) according to manufacturer’s instructions. In brief, 1 x 106 J-774A.1 macrophages infected with Dd8 strain of L. donovani (1:10 ratio) were incubated with test compounds at various concentrations either alone or in combination in 6well plates in duplicate. Culture supernatants were collected after 48 h and analyzed for Th1/Th2 cytokines (IL-12, TNF-α, IL-10 and TGF-β) level as described previously (Shakya et al., 2012). The optical densities of the test samples were measured at 450 nm with λ correction 570 nm in a microplate reader. 2.4. Assessment of nitric oxide (NO) generation The presence of NO in culture supernatant of macrophages was determined by Griess reagent (Shivahare et al., 2014b). Cells from different experimental groups were incubated for 48 h with tested compounds in 5% CO2 incubator at 37°C. Lipopolysaccharide (LPS, 1 μg/mL) was used as a standard stimulant. The nitrite concentration in the macrophages culture supernatant was calculated by comparing with a standard curve generated by sequentially diluting sodium nitrite in deionized water. The intensity of the color developed was measured spectrophotometrically with the blank well set as zero. (Garg et al., 2006).
2.5. Estimation of reactive oxygen species (ROS) level
Leishmania infected macrophages (except uninfected normal and infected control macrophages) were treated with test compounds either alone or in combination were incubated at 37°C in a CO2 incubator for 48 h. After incubation, generation of ROS from macrophages in the presence of compounds was identified using 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA), a non-fluorescent dye that is converted to a fluorescent dye dichlorofluorescein (DCF) in the presence of free radicals and fluorescence of DCFDA was measured by flow cytometry (Walrand et al., 2003). 2.6. Study of Phagocytic activity A flow-cytometry based method was used to study the phagocytic activity of macrophages (Sharma et al., 2004). Murine J-774A.1 macrophages (1x106 cells/mL) infected with promastigotes (1:10 ratio of macrophages: promastigote) were incubated with test compounds in 24 well tissue culture plates at 37°C in a CO2 incubator for 48 h. After incubation, the nonadherent macrophages were removed by washing and incubated with 10 µM fluoroscein isothiocynate (FITC) – labeled E. coli (1:10 ratio) except control wells for 30 min at 37°C. After incubation, excess non-phagocytized bacteria were removed by washing. The cells were collected in tubes and phagocytosis observed by FACS Calibur with FL1 UV band pass filter (excitation at 488 nm and emission at 510 to 513 ± 30 nm). Results were represented as phagocytic index which is the ratio of mean OD of stimulated cells to mean OD of un-stimulated cells.
2.7. Statistical analysis
Data are the representative of three independent experiments and represents mean ± standard deviation. The results were analyzed by one-way ANOVA followed by Tukey’s test using GraphPad Prism (version 5.0) software. 3. Results and Discussion 3.1. In vitro dose standardization of leptin in J-774A.1 macrophages In order to standardize the dose of immunomodulator leptin, we have assessed the hallmark host protective Th1 cytokines (IL-12 and TNF-α) and parasite protective Th2 cytokines (IL-10 and TGF-β) along with the estimation of nitric oxide (NO) generation by macrophages. We have incubated J-774A.1 macrophages with six concentrations of leptin (ranging from 1.25 µg/mL to 20 µg/mL) in a 37°C CO2 incubator. After incubation with leptin for 48 h, we have taken culture supernatant and processed for the assessment of cytokine levels and NO generation. We investigated that most of the concentrations of leptin were significantly (*p<0.05, **p<0.01, ***p<0.001) elevated the levels of IL-12 and TNF-α with maximum elevation (2.3- and 3.7-fold increase respectively in comparison to normal macrophages) in both 15 µg/mL and 20 µg/mL concentrations (Figure 1). Similar to Th1 cytokines, maximum nitric oxide generation by the cells was observed when incubated with 15 µg/mL and 20 µg/mL concentrations of leptin. Contrary to elevation of Th1 cytokines and NO, the level of IL-10 was significantly (*p<0.05, **p<0.01, ***p<0.001) decreased in most of the concentrations with maximum reduction in the groups of 15 µg/mL and 20 µg/mL concentrations of leptin (Figure 1). The level of TGF-β cytokine was also decreased in all the treated groups but the reduction was not significant. 3.2. In vitro antileishmanial efficacy of leptin and miltefosine We demonstrated that leptin at the concentrations of 15 µg/mL and 20 µg/mL significantly elevated the levels of IL-12, TNF-α and NO and also reduced the level of IL-10 than that of normal macrophages. Further, we checked the antileishmanial efficacy of leptin at the same
concentrations (1.25 µg/mL - 20 µg/mL), in which dose of leptin was standardized. We observed that leptin at 15 µg/mL concentration showed 60.5 % inhibition in parasite burden in Leishmania infected macrophages (macrophage / amastigote system) when compare with the group of infected macrophages (Figure 2). Similarly, at 20 µg/mL, leptin exhibited 61.3 % inhibition in parasite multiplication. Since, both 15 µg/mL and 20 µg/mL concentrations of leptin showed almost comparable potential, we have chosen 15 µg/mL concentration of leptin for combination therapy with lower concentration of miltefosine. In the mean time, we have assessed the IC50 of miltefosine in Leishmania infected murine macrophages. IC50 value of miltefosine against the intracellular amastigote form of Leishmania donovani was found to be 8.4 µM (Figure 2). 3.3. Leptin reduces the inhibitory concentration of miltefosine After finding out the best dose of leptin, we have tested six concentrations (1.56-50 µM) of standard drug miltefosine alone and in combination with constant 15 µg/mL concentration of leptin against intramacrophagic amastigotes. Results are presented in Figure 3. Combination of miltefosine with leptin has shown enhanced antileishmanial efficacy at each concentration tested as compared to miltefosine alone. When we tested miltefosine alone, it displayed antiamastigote activity at the IC50 value of 7.5 µM; however, in combination with leptin, the IC50 concentration of miltefosine was reduced by two folds from 7.5 µM to 3.7 µM. Results indicate that reduction in parasitic burden in the infected macrophages by short dose of miltefosine is might be due to the synergistic effect of leptin. Further, we investigated the immune responses generated during combination therapy using the combination dose of leptin (15 µg/mL) and miltefosine (4 µM) along with leptin (15 µg/mL), miltefosine (8 µM) and miltefosine (4 µM) alone groups.
3.4. Combination of leptin and miltefosine induces Th1 cytokines and nitric oxide production We have explored the basic immune parameters in L. donovani infected macrophages that were treated with leptin (15 µg/mL) alone and in combination with miltefosine (4 µM). We investigated the level of Th1/ Th2 cytokines and NO generation in culture supernatant of uninfected, infected/control and infected/treated macrophages. The level of IL-12 and TNF-α cytokines were significantly (***p<0.001) increased in all the treated group as compared to infected control macrophages with maximum elevation (7.7 and 8.1 fold, respectively) in leptinmiltefosine combination treated group (Figure 4). This was even greater as compared to the group treated with 8 µM concentration (IC50 dose) of miltefosine (5.6 and 4.1 fold higher production of IL-12 and TNF-α than infected control, respectively). Similar to Th1 cytokines, treatment of Leishmania infected macrophages with leptin plus low concentration of miltefosine led to a maximum (***p<0.001) generation of nitric oxide (15.43 µM) at 48 h post treatment which was 6.6 fold higher than infected control macrophages (2.33 µM) (Figure 4). Contrary to these observations, Leishmania infected control macrophages showed a remarkable increase in the level of IL-10 and TGF-β cytokines which were significantly (***p<0.001) decreased in all treated groups at same time point (Figure 4). Here too, we observed maximum reduction in leptin plus miltefosine treated macrophages. Taken collectively, increased level of Th1 cytokines and NO and reduced level of Th2 cytokines in leptin plus miltefosine treated macrophages emphasize that low concentration of miltefosine along with 15 µg/mL leptin has the potential to induce better host protective cellular immune responses than that of leptin alone or the higher concentration of miltefosine alone. 3.5. Augmentation of reactive oxygen species during combination therapy Nitric oxide (NO) and reactive oxygen species/intermediates are two effective macrophagederived microbicidal molecules that are essential in controlling Leishmania infection. In addition
to nitric oxide, we have also assessed the generation of intracellular ROS in the same groups of macrophages that were used for the estimation of Th1/Th2 cytokines and NO generation and the data are presented in Figure 5. Leishmania infected macrophages treated with the combination of leptin and miltefosine, showed 1.2-fold induction in ROS generation compared with corresponding infected control macrophages after 48 h of incubation. 3.6. Phagocytic activity of macrophages during combination therapy Phagocytosis is an important mechanism of macrophages for defense against various infections. In this study, we wanted to explore the potential of leptin in inducing the phagocytic ability of Leishmania infected macrophages. From the results (Figure 6), it is evident that leptin (15 µg/mL) alone and miltefosine (4 and 8 µM) alone are not proficient to the induction of phagocytic activity (phagocytic index 21.26 ± 1.90, 20.31 ± 2.10 and 22.53 ± 2.30 respectively) of macrophages infected with Leishmania parasite than that of infected control macrophages (phagocytic index 19.70 ± 1.90) although in the combination group of leptin and miltefosine (4 µM), the significant (**p<0.01) increase in phagocytic activity (phagocytic index 28.55 ± 2.10) was observed. 4. Conclusion Activation of protective responses in immune cells with the help of immunomodulators is critical for overcoming immunosuppression. In the present study, we have shown that leptin in combination with low concentration of miltefosine enhances the host protective immunological responses that help in reducing the parasitic load in murine macrophages. These results demonstrated that inhibition of parasite burden during combination therapy is associated with the induction of cell-mediated immune responses including elevation of Th1 cytokines, IL-12 and TNF-α and subsequent nitric oxide generation. Our results showed that leptin reduces the effective concentration (IC50 dose) of miltefosine by two-folds from 7.5
µM to 3.7 µM. Leptin, a 16 kDa non-glycosylated polypeptide is well known inducer of proinflammatory cytokine, TNF-α and it is also reflected in our results that the combination group induces the secretion of TNF-α (8.1 fold higher) from the macrophages when compare with infected control macrophages. Apart from TNF-α induction, combination group also induces the higher level of IL-12 than that of leptin alone or miltefosine alone group which subsequently stimulates the synthesis of higher amount of nitric oxide and reactive oxygen intermediates. It is already reported that leptin induces the phagocytic ability of macrophages (Dayakar et al., 2011); similar observations were recorded in our study, although, induction of macrophages for increased phagocytic activity was not significant in leptin (15 µg/mL) alone treated group. In human body, leptin regulates thymic homeostasis, participates in host immunity and also act as an adipokine to involve in cytokine induction by T lymphocytes (Conde et al., 2014). The previously published studies indicate that leptin may activate T lymphocytes toward a pro-inflammatory Th1 phenotype via the activation of JAK-STAT (janus associated kinasessignal transducers and activators of transcription) signaling pathway (Martin-Romero et al., 2000). The effect of leptin-driven skewing of T cells toward a Th1 type response seems to be mediated by the stimulation of the synthesis of TNF-α, IL-2, IL-12 and IFN-γ and by the inhibition of IL-4 and IL-10 release (Loffreda et al., 1998; Lord et al., 1998; Martin-Romero et al., 2000). Till to date, no reports have been published which determined the levels of leptin during active VL infection in human. Matarese et al reported that normal leptin levels maintains the Th1/Th2 balance, while if leptin level become low due to nutritional deficiency, reduced levels of Th1 type immune responses and increased levels of Th2 type responses were observed (Matarese et al., 2002). However, it is hypothesized that leptin levels may diminish in VL patients due to malnutrition which lead to the impaired cell mediated immunity against Leishmania infection (Dayakar et al., 2011). Overall, it can be
concluded that leptin reduces the effective concentration of miltefosine and generates elevated level of host protective responses to suppress Leishmania parasite when given with the low dose of miltefosine. Diminution of parasitic load in murine macrophages by lower concentration of miltefosine might be due to the effect of heightened immune responses generated by leptin. These promising results in in vitro macrophage / amastigote system persuade exploring this combination in experimental in vivo animal model of visceral leishmaniasis. Acknowledgements The authors thank Head, Department of Pathology, KGMU, Lucknow and Director, CSIRCentral Drug Research Institute, Lucknow, for encouragement and facilities. R.S. is grateful to Indian Council of Medical Research, New Delhi, for financial support in the form of senior research fellowship. The L. donovani (DD8) promastigotes were originally procured from CSIR- CDRI, Lucknow, India. The structure of leptin in graphical abstract is taken from the web (http://en.wikipedia.org/wiki/Leptin) with due acknowledgement to the owner.
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Figure captions with legends Fig. 1. In vitro dose standardization of leptin (LPT) in J-774A.1 macrophages. Murine macrophages were incubated with the various concentrations of leptin as shown in figure legends. The level of Th1/Th2 cytokines and nitric oxide production were estimated in the culture supernatant by ELISA and Griess assay, respectively. Data are the representative of three independent experiments and represents mean ± standard deviation. The significance between
different experimental groups was calculated by one way ANOVA followed by Tukey’s post test using graph pad Prism (version 5.0). [Significance: Normal vs treated groups; *p<0.05, **p<0.01, ***p<0.001; $non-significant].
Fig. 2. In vitro antileishmanial efficacy of leptin and miltefosine against intracellular amastigote form of Leishmania donovani. The experiment for the assessment of antileishmanial efficacy was performed using Leishmania promastigotes infected J-774A.1 macrophages treated with various concentrations of leptin and miltefosine as shown in figure 2. Inhibition in parasite multiplication was assessed by microscopic observation followed by Giemsa’s staining. Data are the representative of three independent experiments and represents mean ± standard deviation. The percent inhibition was determined by comparison of treated groups with untreated group and calculated by the formula given in materials and methods section. The IC50 value of miltefosine was calculated by the non-linear regression analysis of the concentration response curve using the four parameter Hill equations.
Fig. 3. Assessment of antileishmanial efficacy during combination therapy with leptin and miltefosine. The experiment was performed using Leishmania promastigotes infected J-774A.1 macrophages treated with various concentrations of miltefosine alone and in combination with 15 µg/mL concentration of leptin. Inhibition of parasite multiplication was assessed by microscopic observations followed by Giemsa’s staining. Data are the representative of three independent experiments and represents mean ± standard deviation. The percent inhibition was determined by comparison of treated groups with untreated group and calculated by the formula given in materials and methods section. The IC50 value of miltefosine was calculated by the nonlinear regression analysis of the concentration response curve using the four parameter Hill equations.
Fig. 4. Effect of leptin (LPT) and miltefosine (MLT) combination therapy on Th1/Th2 cytokine response and NO generation. Murine macrophage J-774A.1 cells were infected with L. donovani and treated with leptin alone, miltefosine alone and combination of leptin plus miltefosine as described in figure legends. The level of Th1 (IL-12 and TNF-α) /Th2 (IL-10 and TGF-β) cytokines and nitric oxide production was estimated in the culture supernatant by ELISA and Griess assay, respectively. Three independent experiments were done and each bar represents pooled data (mean ± SD). The significance between different experimental groups was calculated by one way ANOVA followed by Tukey’s post test using graph pad Prism (version 5.0). [Significance: Infected control vs normal and treated groups, $non-significant; **p<0.01, ***p<0.001].
Fig. 5. Effect of combination therapy on the production of reactive oxygen species. Leishmania infected macrophages (J-774A.1 cells) were treated with leptin (LPT) alone, miltefosine (MLT) alone and combination of leptin plus miltefosine as described in figure legends. The cells of normal, infected and treated groups were collected and processed for flow cytometric analysis for the estimation of reactive oxygen species using H2DCFDA dye as described in materials and methods section. Mean ± SD were calculated by the comparison of normal and treated groups to its infected counterparts. The data are representative of three independent experiments. The significance between different experimental groups was calculated by one way ANOVA followed by Tukey’s post test using graph pad Prism (version 5.0). [Significance: Infected control vs normal and treated groups, $non-significant; *p<0.05].
Fig. 6. Effect of combination therapy on the phagocytic activity of murine macrophages. Leishmania infected macrophages (J-774A.1) were treated with leptin (LPT) alone, miltefosine (MLT) alone and combination of leptin plus miltefosine as described in figure legend. After 48 h
incubation, macrophages (except un-stimulated control) were stimulated with fluorescein isothiocyanate (FITC) - labelled E.coli, incubated for 30 min and processed for flow cytometric analysis. Fluorescence of stimulated and un-stimulated cells of each group compared and phagocytic index was calculated by the formula given in materials and methods section. The data are the representative of three independent experiments. The significance between different experimental groups was calculated by one way ANOVA followed by Tukey’s post test using graph pad Prism (version 5.0). [Significance: Infected control vs normal and treated groups, $
non-significant; **p<0.01].
Figures Fig. 1. In vitro dose standardization of leptin (LPT) in J-774A.1 macrophages
Fig. 2. In vitro antileishmanial efficacy of leptin and miltefosine against intracellular amastigote form of Leishmania donovani
Fig. 3. Assessment of antileishmanial efficacy during combination therapy with leptin and miltefosine
Fig. 4. Effect of leptin (LPT) and miltefosine (MLT) combination therapy on Th1/Th2 cytokine response and NO generation
Fig. 5. Effect of combination therapy on the production of intracellular reactive oxygen species
Fig. 6. Effect of combination therapy on the phagocytic activity of murine macrophages
S0001-706X(15)30045-0 http://dx.doi.org/doi:10.1016/j.actatropica.2015.06.024 ACTROP 3666
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
5-3-2015 19-6-2015 22-6-2015
Please cite this article as: Shivahare, Rahul, Ali, Wahid, Vishwakarma, Preeti, Natu, S.M., Puri, Sunil K., Gupta, Suman, Leptin Augments Protective Immune Responses in Murine Macrophages and Enhances Potential of Miltefosine against Experimental Visceral Leishmaniasis.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2015.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Leptin Augments Protective Immune Responses in Murine Macrophages and Enhances Potential of Miltefosine against Experimental Visceral Leishmaniasis Rahul Shivaharea*, Wahid Alia, Preeti Vishwakarmab, S.M. Natua, Sunil K. Purib, Suman Guptab a
b
Department of Pathology, King George’s Medical University, Lucknow- 226 003 (India)
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow- 226 031 (India)
*Corresponding author Department of Pathology, King George’s Medical University, Lucknow- 226 003 (India) E-mail addresses: [email protected]; [email protected] Phone: +91 522 2257580, +91 9935300350; Fax: +91 522 2257580
Highlights
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Leptin reduces effective concentration of miltefosine by two-folds.
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Leptin induces host macrophages for the activation of Th1 biased immune responses.
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Leptin when combining with miltefosine boosted up host immunity.
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Leptin plus miltefosine heightened the phagocytic activity of macrophages.
Abstract Adverse side effects and drug resistance issues are the two most important drawbacks which influence the widespread use of existing antileishmanial drugs. Use of immune stimulating agent with standard antileishmanial might be helpful to minimize the toxic effect of drug, shorten the dose regimen and delay the emergence of resistance. In the present study, we explored the in vitro immunomodulatory potential of an immunomodulator, leptin with lower concentration of standard drug, miltefosine. The level of Th1/Th2 cytokines, production of nitric oxide and reactive oxygen species and phagocytic activity were assessed by ELISA, Griess reaction and flow cytometric analysis, respectively. Leptin at a concentration of 15 µg/mL showed heightened level of Th1 cytokines and nitric oxide generation from murine macrophages (J-774A.1 cells). Leptin (15 µg/mL) also reduces the effective concentration of miltefosine by 2-folds from 7.5 µM to 3.7 µM. When given in conjunction with lower concentration of miltefosine (4 µM), leptin (15 µg/mL) was significantly (***p<0.001) elevated the level of IL-12 (7.7 fold), TNF-α (8.1 fold) and nitric oxide (6.6 fold) along with markedly (***p<0.001) suppressed level of IL-10 and TGF-β when compared with untreated infected macrophages. Leptin plus miltefosine also induces the phagocytic ability (**p<0.01) of macrophages in comparison to leptin alone and miltefosine alone treated groups. These finding illustrate that leptin activates host macrophages to generate protective immune response for the successful elimination of Leishmania parasite at lower concentration of miltefosine and has potential for further exploration in experimental animal model of visceral leishmaniasis (VL). Keywords: Leishmania donovani; J-774.A1 cells; Leptin; Miltefosine; Combination therapy; Immune response
1. Introduction Visceral leishmaniasis (VL) or black fever (kala-azar in Hindi) is a neglected infectious disease caused by the protozoan parasite of the genus Leishmania in tropical and subtropical regions particularly in the Indian subcontinent, East Africa and South America (Guerin et al., 2002). VL threatens 200 million people in 62 countries with an estimated 5,00,000 new cases and 60,000 deaths each year (Desjeux, 2004). This systemic infection affects the poorest people mainly in rural areas and it is a main public health problem in Indian sub-continent with more than 60% of global cases (Alvar et al., 2012). VL is potentially fatal disease in which parasite manipulates the normal immune function of the host for the survival and reside within the macrophages of spleen, liver and bone marrow (Stanley and Engwerda, 2007; Murray et al., 2003). Leishmania infected host macrophages are incapable to produce inflammatory responses which involves production of tumor necrosis factor (TNF)-α, interleukin (IL)-12- dependent production of interferon (IFN)-γ and subsequent generation of nitric oxide (NO) (Olivier et al., 2005). In addition, Leishmania parasite can stimulate the macrophages for the production of immunosuppressive molecules, IL-10, transforming growth factor (TGF)-β and prostaglandin E2 (PGE2). These molecules directly or indirectly distort normal immune function and favor parasite survival within the host macrophages (Olivier et al., 2005). The co-infection of HIV is exacerbated the morbidity and mortality rate in immuno-compromised VL patients (Tiuman et al., 2011). Currently, antileishmanial chemotherapy relies on handful number of drugs including pentavalent antimonials, amphotericin B and its formulations, paromomycin and oral drug miltefosine. These all therapeutic options are unaffordable to poor patients, have adverse side effects and drug resistance issues (Alvar et al., 2006; Croft et al., 2006). None of the medicaments are effective in all cases of VL infection. In recent years, several reports indicated that combinations of existing antileishmanial drug with another antileishmanial agent or with
immunomodulators are advantageous over monotherapy as they minimize the toxic effect of drug, shorten the dose regimen and delay or prevent the emergence of resistance (Seifert and Croft, 2006; Musa et al., 2012; Omollo et al., 2011; Olliaro, 2010). Since, diminish immune function is responsible for the progression of Leishmania infection in host, immunomodulator along with chemotherapy might be beneficial to boost up host immune cells to generate protective immune response for the successful treatment of VL at short dose regimen. Some potential immunomodulators such as imiquimod with paromomycin (El-On et al., 2007), tamoxifen with amphotericin B (Trinconi et al., 2014) and CpG-ODNs with miltefosine (Sane et al., 2010; Shivahare et al., 2014a) have been explored for the successful treatment of cutaneous and visceral forms of leishmaniasis. In the present study, we have demonstrated a new combination of a potent immune stimulating agent, leptin and oral drug, miltefosine. Leptin is an adipocyte derived hormone which has pro-inflammatory effects on T cell populations. It is reported that leptin stimulates macrophages and natural killer cells for the production of pro-inflammatory Th1 cytokines such as IL-2, IFN-γ, TNF-α and IL-18, with resultant decreased production of the Th2 cytokines, IL-4, IL-5 and IL-10 (Lord et al., 1998; Martin-Romero et al., 2000). Considering these interesting reports, we explored the effects of combination therapy in parasite survival, alterations in immunological and biochemical parameters viz. Th1/Th2 cytokines, production of NO and reactive oxygen species (ROS) and phagocytic activity in Leishmania infected murine macrophages (J-774A.1 cell line) when incubated/ treated with leptin alone or in combination with lower concentration of miltefosine.
2. Materials and Methods 2.1. Parasite, cell culture and compounds
The WHO reference strain of Leishmania donovani (MHOM/IN/80/Dd8) was maintained in the laboratory as promastigotes in vitro in M-199 medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS) at 24ºC ± 2ºC. Murine macrophage cell line (J-774A.1 cells) obtained from National Centre for Cell Sciences, Pune (India) was cultured in RPMI-1640 medium (Sigma-Aldrich, USA) supplemented with 10% heat inactivated FBS at 37°C in a 5% CO2 incubator. Leptin (Peprotech, USA) and miltefosine (Synphabase AG, Switzerland) were dissolved in deionized water and phosphate buffer saline (PBS) respectively prior to in vitro experiments. 2.2. In vitro assessment in macrophage- amastigote system Mouse macrophages (J-774A.1 cells) (2 x 104/mL) were seeded in complete RPMI medium and layered in 16 well chamber slides (Nunc) under a final volume of 100 µL/well and allowed to adhere overnight at 37°C in a 5% CO2-95% air mixture. Macrophages were infected with stationary phase promastigotes at a macrophage-promastigote ratio of 1:10 on day 2. Infected culture was maintained at 37°C in a 5% CO2 incubator. Promastigotes were phagocytized by the macrophages and inside the phagolysosomes, they were transformed into amastigotes (nonmotile form). On day 3, test compounds were prepared in complete RPMI medium and added at two fold dilution in complete medium at different concentrations after replacing the previous medium and the slides were incubated at 37°C in a 5% CO2 incubator. Compounds containing medium were changed on day 5 and slides were incubated at 37°C in a 5% CO2 incubator. Finally slides were fixed with 100% methanol and stained with 10% Giemsa stain for 45 min on day 6 (Seifert et al., 2006; Gupta et al., 2002). The number of amastigotes per 500 cell nuclei was counted in each well and the parasitic burden is expressed in terms of the number of amastigote per 100 cell nuclei. Drug activity (percent inhibition) was determined by comparing amastigote count of treated and untreated wells by the general formula:
Percent Inhibition (PI) =
Where, N is average number of amastigotes per 100 cell nuclei of untreated well and n is average number of amastigotes per 100 cell nuclei of treated well. 2.3. Estimation of cytokine levels The level of various cytokines secreted by murine macrophages was measured using a BD OptEIA ELISA kit (BD Biosciences, USA) according to manufacturer’s instructions. In brief, 1 x 106 J-774A.1 macrophages infected with Dd8 strain of L. donovani (1:10 ratio) were incubated with test compounds at various concentrations either alone or in combination in 6well plates in duplicate. Culture supernatants were collected after 48 h and analyzed for Th1/Th2 cytokines (IL-12, TNF-α, IL-10 and TGF-β) level as described previously (Shakya et al., 2012). The optical densities of the test samples were measured at 450 nm with λ correction 570 nm in a microplate reader. 2.4. Assessment of nitric oxide (NO) generation The presence of NO in culture supernatant of macrophages was determined by Griess reagent (Shivahare et al., 2014b). Cells from different experimental groups were incubated for 48 h with tested compounds in 5% CO2 incubator at 37°C. Lipopolysaccharide (LPS, 1 μg/mL) was used as a standard stimulant. The nitrite concentration in the macrophages culture supernatant was calculated by comparing with a standard curve generated by sequentially diluting sodium nitrite in deionized water. The intensity of the color developed was measured spectrophotometrically with the blank well set as zero. (Garg et al., 2006).
2.5. Estimation of reactive oxygen species (ROS) level
Leishmania infected macrophages (except uninfected normal and infected control macrophages) were treated with test compounds either alone or in combination were incubated at 37°C in a CO2 incubator for 48 h. After incubation, generation of ROS from macrophages in the presence of compounds was identified using 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA), a non-fluorescent dye that is converted to a fluorescent dye dichlorofluorescein (DCF) in the presence of free radicals and fluorescence of DCFDA was measured by flow cytometry (Walrand et al., 2003). 2.6. Study of Phagocytic activity A flow-cytometry based method was used to study the phagocytic activity of macrophages (Sharma et al., 2004). Murine J-774A.1 macrophages (1x106 cells/mL) infected with promastigotes (1:10 ratio of macrophages: promastigote) were incubated with test compounds in 24 well tissue culture plates at 37°C in a CO2 incubator for 48 h. After incubation, the nonadherent macrophages were removed by washing and incubated with 10 µM fluoroscein isothiocynate (FITC) – labeled E. coli (1:10 ratio) except control wells for 30 min at 37°C. After incubation, excess non-phagocytized bacteria were removed by washing. The cells were collected in tubes and phagocytosis observed by FACS Calibur with FL1 UV band pass filter (excitation at 488 nm and emission at 510 to 513 ± 30 nm). Results were represented as phagocytic index which is the ratio of mean OD of stimulated cells to mean OD of un-stimulated cells.
2.7. Statistical analysis
Data are the representative of three independent experiments and represents mean ± standard deviation. The results were analyzed by one-way ANOVA followed by Tukey’s test using GraphPad Prism (version 5.0) software. 3. Results and Discussion 3.1. In vitro dose standardization of leptin in J-774A.1 macrophages In order to standardize the dose of immunomodulator leptin, we have assessed the hallmark host protective Th1 cytokines (IL-12 and TNF-α) and parasite protective Th2 cytokines (IL-10 and TGF-β) along with the estimation of nitric oxide (NO) generation by macrophages. We have incubated J-774A.1 macrophages with six concentrations of leptin (ranging from 1.25 µg/mL to 20 µg/mL) in a 37°C CO2 incubator. After incubation with leptin for 48 h, we have taken culture supernatant and processed for the assessment of cytokine levels and NO generation. We investigated that most of the concentrations of leptin were significantly (*p<0.05, **p<0.01, ***p<0.001) elevated the levels of IL-12 and TNF-α with maximum elevation (2.3- and 3.7-fold increase respectively in comparison to normal macrophages) in both 15 µg/mL and 20 µg/mL concentrations (Figure 1). Similar to Th1 cytokines, maximum nitric oxide generation by the cells was observed when incubated with 15 µg/mL and 20 µg/mL concentrations of leptin. Contrary to elevation of Th1 cytokines and NO, the level of IL-10 was significantly (*p<0.05, **p<0.01, ***p<0.001) decreased in most of the concentrations with maximum reduction in the groups of 15 µg/mL and 20 µg/mL concentrations of leptin (Figure 1). The level of TGF-β cytokine was also decreased in all the treated groups but the reduction was not significant. 3.2. In vitro antileishmanial efficacy of leptin and miltefosine We demonstrated that leptin at the concentrations of 15 µg/mL and 20 µg/mL significantly elevated the levels of IL-12, TNF-α and NO and also reduced the level of IL-10 than that of normal macrophages. Further, we checked the antileishmanial efficacy of leptin at the same
concentrations (1.25 µg/mL - 20 µg/mL), in which dose of leptin was standardized. We observed that leptin at 15 µg/mL concentration showed 60.5 % inhibition in parasite burden in Leishmania infected macrophages (macrophage / amastigote system) when compare with the group of infected macrophages (Figure 2). Similarly, at 20 µg/mL, leptin exhibited 61.3 % inhibition in parasite multiplication. Since, both 15 µg/mL and 20 µg/mL concentrations of leptin showed almost comparable potential, we have chosen 15 µg/mL concentration of leptin for combination therapy with lower concentration of miltefosine. In the mean time, we have assessed the IC50 of miltefosine in Leishmania infected murine macrophages. IC50 value of miltefosine against the intracellular amastigote form of Leishmania donovani was found to be 8.4 µM (Figure 2). 3.3. Leptin reduces the inhibitory concentration of miltefosine After finding out the best dose of leptin, we have tested six concentrations (1.56-50 µM) of standard drug miltefosine alone and in combination with constant 15 µg/mL concentration of leptin against intramacrophagic amastigotes. Results are presented in Figure 3. Combination of miltefosine with leptin has shown enhanced antileishmanial efficacy at each concentration tested as compared to miltefosine alone. When we tested miltefosine alone, it displayed antiamastigote activity at the IC50 value of 7.5 µM; however, in combination with leptin, the IC50 concentration of miltefosine was reduced by two folds from 7.5 µM to 3.7 µM. Results indicate that reduction in parasitic burden in the infected macrophages by short dose of miltefosine is might be due to the synergistic effect of leptin. Further, we investigated the immune responses generated during combination therapy using the combination dose of leptin (15 µg/mL) and miltefosine (4 µM) along with leptin (15 µg/mL), miltefosine (8 µM) and miltefosine (4 µM) alone groups.
3.4. Combination of leptin and miltefosine induces Th1 cytokines and nitric oxide production We have explored the basic immune parameters in L. donovani infected macrophages that were treated with leptin (15 µg/mL) alone and in combination with miltefosine (4 µM). We investigated the level of Th1/ Th2 cytokines and NO generation in culture supernatant of uninfected, infected/control and infected/treated macrophages. The level of IL-12 and TNF-α cytokines were significantly (***p<0.001) increased in all the treated group as compared to infected control macrophages with maximum elevation (7.7 and 8.1 fold, respectively) in leptinmiltefosine combination treated group (Figure 4). This was even greater as compared to the group treated with 8 µM concentration (IC50 dose) of miltefosine (5.6 and 4.1 fold higher production of IL-12 and TNF-α than infected control, respectively). Similar to Th1 cytokines, treatment of Leishmania infected macrophages with leptin plus low concentration of miltefosine led to a maximum (***p<0.001) generation of nitric oxide (15.43 µM) at 48 h post treatment which was 6.6 fold higher than infected control macrophages (2.33 µM) (Figure 4). Contrary to these observations, Leishmania infected control macrophages showed a remarkable increase in the level of IL-10 and TGF-β cytokines which were significantly (***p<0.001) decreased in all treated groups at same time point (Figure 4). Here too, we observed maximum reduction in leptin plus miltefosine treated macrophages. Taken collectively, increased level of Th1 cytokines and NO and reduced level of Th2 cytokines in leptin plus miltefosine treated macrophages emphasize that low concentration of miltefosine along with 15 µg/mL leptin has the potential to induce better host protective cellular immune responses than that of leptin alone or the higher concentration of miltefosine alone. 3.5. Augmentation of reactive oxygen species during combination therapy Nitric oxide (NO) and reactive oxygen species/intermediates are two effective macrophagederived microbicidal molecules that are essential in controlling Leishmania infection. In addition
to nitric oxide, we have also assessed the generation of intracellular ROS in the same groups of macrophages that were used for the estimation of Th1/Th2 cytokines and NO generation and the data are presented in Figure 5. Leishmania infected macrophages treated with the combination of leptin and miltefosine, showed 1.2-fold induction in ROS generation compared with corresponding infected control macrophages after 48 h of incubation. 3.6. Phagocytic activity of macrophages during combination therapy Phagocytosis is an important mechanism of macrophages for defense against various infections. In this study, we wanted to explore the potential of leptin in inducing the phagocytic ability of Leishmania infected macrophages. From the results (Figure 6), it is evident that leptin (15 µg/mL) alone and miltefosine (4 and 8 µM) alone are not proficient to the induction of phagocytic activity (phagocytic index 21.26 ± 1.90, 20.31 ± 2.10 and 22.53 ± 2.30 respectively) of macrophages infected with Leishmania parasite than that of infected control macrophages (phagocytic index 19.70 ± 1.90) although in the combination group of leptin and miltefosine (4 µM), the significant (**p<0.01) increase in phagocytic activity (phagocytic index 28.55 ± 2.10) was observed. 4. Conclusion Activation of protective responses in immune cells with the help of immunomodulators is critical for overcoming immunosuppression. In the present study, we have shown that leptin in combination with low concentration of miltefosine enhances the host protective immunological responses that help in reducing the parasitic load in murine macrophages. These results demonstrated that inhibition of parasite burden during combination therapy is associated with the induction of cell-mediated immune responses including elevation of Th1 cytokines, IL-12 and TNF-α and subsequent nitric oxide generation. Our results showed that leptin reduces the effective concentration (IC50 dose) of miltefosine by two-folds from 7.5
µM to 3.7 µM. Leptin, a 16 kDa non-glycosylated polypeptide is well known inducer of proinflammatory cytokine, TNF-α and it is also reflected in our results that the combination group induces the secretion of TNF-α (8.1 fold higher) from the macrophages when compare with infected control macrophages. Apart from TNF-α induction, combination group also induces the higher level of IL-12 than that of leptin alone or miltefosine alone group which subsequently stimulates the synthesis of higher amount of nitric oxide and reactive oxygen intermediates. It is already reported that leptin induces the phagocytic ability of macrophages (Dayakar et al., 2011); similar observations were recorded in our study, although, induction of macrophages for increased phagocytic activity was not significant in leptin (15 µg/mL) alone treated group. In human body, leptin regulates thymic homeostasis, participates in host immunity and also act as an adipokine to involve in cytokine induction by T lymphocytes (Conde et al., 2014). The previously published studies indicate that leptin may activate T lymphocytes toward a pro-inflammatory Th1 phenotype via the activation of JAK-STAT (janus associated kinasessignal transducers and activators of transcription) signaling pathway (Martin-Romero et al., 2000). The effect of leptin-driven skewing of T cells toward a Th1 type response seems to be mediated by the stimulation of the synthesis of TNF-α, IL-2, IL-12 and IFN-γ and by the inhibition of IL-4 and IL-10 release (Loffreda et al., 1998; Lord et al., 1998; Martin-Romero et al., 2000). Till to date, no reports have been published which determined the levels of leptin during active VL infection in human. Matarese et al reported that normal leptin levels maintains the Th1/Th2 balance, while if leptin level become low due to nutritional deficiency, reduced levels of Th1 type immune responses and increased levels of Th2 type responses were observed (Matarese et al., 2002). However, it is hypothesized that leptin levels may diminish in VL patients due to malnutrition which lead to the impaired cell mediated immunity against Leishmania infection (Dayakar et al., 2011). Overall, it can be
concluded that leptin reduces the effective concentration of miltefosine and generates elevated level of host protective responses to suppress Leishmania parasite when given with the low dose of miltefosine. Diminution of parasitic load in murine macrophages by lower concentration of miltefosine might be due to the effect of heightened immune responses generated by leptin. These promising results in in vitro macrophage / amastigote system persuade exploring this combination in experimental in vivo animal model of visceral leishmaniasis. Acknowledgements The authors thank Head, Department of Pathology, KGMU, Lucknow and Director, CSIRCentral Drug Research Institute, Lucknow, for encouragement and facilities. R.S. is grateful to Indian Council of Medical Research, New Delhi, for financial support in the form of senior research fellowship. The L. donovani (DD8) promastigotes were originally procured from CSIR- CDRI, Lucknow, India. The structure of leptin in graphical abstract is taken from the web (http://en.wikipedia.org/wiki/Leptin) with due acknowledgement to the owner.
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Figure captions with legends Fig. 1. In vitro dose standardization of leptin (LPT) in J-774A.1 macrophages. Murine macrophages were incubated with the various concentrations of leptin as shown in figure legends. The level of Th1/Th2 cytokines and nitric oxide production were estimated in the culture supernatant by ELISA and Griess assay, respectively. Data are the representative of three independent experiments and represents mean ± standard deviation. The significance between
different experimental groups was calculated by one way ANOVA followed by Tukey’s post test using graph pad Prism (version 5.0). [Significance: Normal vs treated groups; *p<0.05, **p<0.01, ***p<0.001; $non-significant].
Fig. 2. In vitro antileishmanial efficacy of leptin and miltefosine against intracellular amastigote form of Leishmania donovani. The experiment for the assessment of antileishmanial efficacy was performed using Leishmania promastigotes infected J-774A.1 macrophages treated with various concentrations of leptin and miltefosine as shown in figure 2. Inhibition in parasite multiplication was assessed by microscopic observation followed by Giemsa’s staining. Data are the representative of three independent experiments and represents mean ± standard deviation. The percent inhibition was determined by comparison of treated groups with untreated group and calculated by the formula given in materials and methods section. The IC50 value of miltefosine was calculated by the non-linear regression analysis of the concentration response curve using the four parameter Hill equations.
Fig. 3. Assessment of antileishmanial efficacy during combination therapy with leptin and miltefosine. The experiment was performed using Leishmania promastigotes infected J-774A.1 macrophages treated with various concentrations of miltefosine alone and in combination with 15 µg/mL concentration of leptin. Inhibition of parasite multiplication was assessed by microscopic observations followed by Giemsa’s staining. Data are the representative of three independent experiments and represents mean ± standard deviation. The percent inhibition was determined by comparison of treated groups with untreated group and calculated by the formula given in materials and methods section. The IC50 value of miltefosine was calculated by the nonlinear regression analysis of the concentration response curve using the four parameter Hill equations.
Fig. 4. Effect of leptin (LPT) and miltefosine (MLT) combination therapy on Th1/Th2 cytokine response and NO generation. Murine macrophage J-774A.1 cells were infected with L. donovani and treated with leptin alone, miltefosine alone and combination of leptin plus miltefosine as described in figure legends. The level of Th1 (IL-12 and TNF-α) /Th2 (IL-10 and TGF-β) cytokines and nitric oxide production was estimated in the culture supernatant by ELISA and Griess assay, respectively. Three independent experiments were done and each bar represents pooled data (mean ± SD). The significance between different experimental groups was calculated by one way ANOVA followed by Tukey’s post test using graph pad Prism (version 5.0). [Significance: Infected control vs normal and treated groups, $non-significant; **p<0.01, ***p<0.001].
Fig. 5. Effect of combination therapy on the production of reactive oxygen species. Leishmania infected macrophages (J-774A.1 cells) were treated with leptin (LPT) alone, miltefosine (MLT) alone and combination of leptin plus miltefosine as described in figure legends. The cells of normal, infected and treated groups were collected and processed for flow cytometric analysis for the estimation of reactive oxygen species using H2DCFDA dye as described in materials and methods section. Mean ± SD were calculated by the comparison of normal and treated groups to its infected counterparts. The data are representative of three independent experiments. The significance between different experimental groups was calculated by one way ANOVA followed by Tukey’s post test using graph pad Prism (version 5.0). [Significance: Infected control vs normal and treated groups, $non-significant; *p<0.05].
Fig. 6. Effect of combination therapy on the phagocytic activity of murine macrophages. Leishmania infected macrophages (J-774A.1) were treated with leptin (LPT) alone, miltefosine (MLT) alone and combination of leptin plus miltefosine as described in figure legend. After 48 h
incubation, macrophages (except un-stimulated control) were stimulated with fluorescein isothiocyanate (FITC) - labelled E.coli, incubated for 30 min and processed for flow cytometric analysis. Fluorescence of stimulated and un-stimulated cells of each group compared and phagocytic index was calculated by the formula given in materials and methods section. The data are the representative of three independent experiments. The significance between different experimental groups was calculated by one way ANOVA followed by Tukey’s post test using graph pad Prism (version 5.0). [Significance: Infected control vs normal and treated groups, $
non-significant; **p<0.01].
Figures Fig. 1. In vitro dose standardization of leptin (LPT) in J-774A.1 macrophages
Fig. 2. In vitro antileishmanial efficacy of leptin and miltefosine against intracellular amastigote form of Leishmania donovani
Fig. 3. Assessment of antileishmanial efficacy during combination therapy with leptin and miltefosine
Fig. 4. Effect of leptin (LPT) and miltefosine (MLT) combination therapy on Th1/Th2 cytokine response and NO generation
Fig. 5. Effect of combination therapy on the production of intracellular reactive oxygen species
Fig. 6. Effect of combination therapy on the phagocytic activity of murine macrophages