Cytokine profile and nitric oxide levels in peritoneal macrophages of BALB/c mice exposed to Fucose-Mannose Ligand of Leishmania infantum combined with Glycyrrhizin

doi:10.21203/rs.3.rs-21571/v3

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Cytokine profile and nitric oxide levels in peritoneal macrophages of BALB/c mice exposed to Fucose-Mannose Ligand of Leishmania infantum combined with Glycyrrhizin

https://doi.org/10.21203/rs.3.rs-21571/v3

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published 20 Jul, 2020

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Background

The Fucose-Mannose Ligand (FML) of Leishmania infantum is a complex glycoprotein which does not elicit adequate immunogenicity in human. In recent years, adjuvant compounds derived from plants have been used for improving the immunogenicity of the vaccines. Glycyrrhizin (GL) is a natural triterpenoid saponin that has known immunomodulatory activities. In the present study, we investigated the effects of a co-treatment with FML and GL on the production of cytokines and nitric oxide ( NO) by macrophages, in vitro .

Methods

Lipopolysaccharide (LPS) stimulated murine peritoneal macrophages were treated with FML (5 μg/ml) of Leishmania infantum and various concentrations of GL (1 μg/ml, or 10 μg/ml or 20 μg/ml). After 48h of treatment, cell culture supernatants were recovered and the levels of TNF-α, IL-10, IL-12p70, and IP-10 were measured by sandwich ELISA and NO concentration by Griess reaction.

Results

Our results indicated that the treatment of activated macrophages with FML plus GL leads to enhanced production of NO, TNF-α, IL-12p70, and reduction of IL-10 levels in comparison with FML treatment alone.

Conclusions

We, therefore, concluded that GL can improve the immunostimulatory effect of FML on macrophages and leads to polarization of them toward an M1-like phenotype.

Parasitology

Fucose-mannose ligand

Glycyrrhizin

Macrophage

Nitric oxide

Visceral Leishmaniasis

Visceral leishmaniasis (VL) or kala-azar, a cosmopolitan vector-borne zoonotic disease, is the most severe and fatal form of leishmaniasis if not diagnosed and treated in time. The disease is caused by parasitic protozoan species of the Leishmania donovani complex and is transmitted by sandflies (1). It is one of the most important parasitic diseases among the many parasitic diseases in Iran (2-5).

In the recent past, the parasites have developed resistance to the existing drugs and also due to lack of effective human vaccine against VL, lead to increase in the incidence of VL (6-9). Different generations of vaccines against the different regions of parasitic antigens have been examined in the hope of finding an appropriate treatment for leishmaniasis or induce protection against the disease with long-term immunity (9-12).

Among the various antigens that serve as targets for VL vaccine design, fucose-mannose ligand (FML) has attracted much attention owing to its excellent immunoprotective properties against experimental VL in several animal models (13). The FML is a glycoprotein antigen which is present both in amastigotes and in motile promastigotes of L. donovani complex. Even though the FML is a potent immunogen in rabbits and dogs (e.g., Leishmune® a vaccine for canine VL consists of FML and saponin) (14), it does not have adequate immunogenicity in human (9, 15).

Previous studies have demonstrated that macrophages play a pivotal role in the outcome of Leishmania infection depending on the type of macrophages; classically activated (M1) macrophages as efficient type against Leishmania parasites or alternatively activated (M2) macrophages as favoring survival and growth of Leishmania parasites (16, 17). In response to different microbial stimuli and immune status of the microenvironment, naive macrophages (M0) differentiate into either M1 or M2 subpopulation with different patterns of cytokine production and distinct properties. Concerning the stimuli that can affect shift macrophages toward M1 or M2 subpopulation, recently we evaluated the immunomodulatory effects of FML on macrophages (18). Our findings showed that although the FML significantly increases nitric oxide (NO), IL-12p70 and IP-10 production in the macrophages, but cannot alter TNF-α production in them (18). The most surprising aspect of this study was that FML significantly increased the production of IL-10, an immunosuppressive cytokine, from macrophages (18).

Glycyrrhizin (GL) is a natural triterpenoid saponin derived from the root of licorice (Glycyrrhiza glabra) that has been associated with numerous pharmacologic effects, including anti-bacterial, anti-inflammatory, anti-oxidant, anti-viral, anti-tumor, hepatoprotective, and immunomodulatory activities (19-22). Stimulation of IL-12 and NO production and suppression of IL-10 production from macrophages (23, 24), augmentation of NK cell activity (23), up regulation of costimulatory molecules on dendritic cells (19), increase in T cell proliferation, reduction of IL-4 production from T cells, and direction of immune response toward Th1 (19), are the most important known immunomodulatory activities of GL.

It is well known that the GL plays an important role in immunomodulatory activities such as stimulation of IL-12 and NO production and suppression of IL-10 production from macrophages (23, 24). The GL also plays a central role in the augmentation of NK cell activity, up-regulation of costimulatory molecules on dendritic cells, increasing of T cells proliferation, reduction of IL-4 production from T cells, and direction of the immune response toward Th1, (19).

With regard to improvements in the production of human VL vaccine by using purified FML and considering the importance of macrophages in protection and control against VL, it seems that characterization of the immunomodulatory effects of combination of FML/GL on macrophages can be useful in finding ways to increase the immunogenicity of FML and vaccine development. Therefore, for the first time, we investigated the effects of FML/GL on production of cytokines and NO by macrophages, in vitro.

Animals

For the experimental studies, a total of seven 6-8 week-old female BALB/c mice were used from Razi Institute (Mashhad, Iran). The mice were fed standard mouse chow ad libitum throughout the study and maintained under the standard conditions according to the protocol. The experimental protocols used in the study were approved by the Animal Ethics Committee of North Khorasan University of Medical Sciences, Bojnurd, Iran (95-914/p).

Leishmania promastigote culture and FML extraction

L. infantum promastigotes (MCAN/IR/07/Moheb-gh) were grown at 26 °C in the brain heart infusion broth (37 g/l; Himedia, India) supplemented with 10% of fetal bovine serum (Gibco, UK), hemin (0.01 g/l) and folic acid (0.02 g/l; Sigma, MO, USA).

The stationary phase growth medium was centrifuged at 6000xg for 10 min to collect the promastigotes. The pellet containing promastigotes was washed with cold phosphate-buffered saline (PBS) and was stored at -20 °C until further analysis. Aqueous extraction of FML was carried out as explained earlier by Foroughi-Parvar et al. (25). Briefly, frozen pellets of the parasite were mixed with cold distilled water and centrifuged at 6000xg for 10 min to collect the supernatant. This step was repeated once again, and both the supernatants were combined and boiled for 15 min at 100 °C. The sample was then centrifuged, and the supernatant was lyophilized and subjected to chromatography by loading 2 ml of lyophilized sample in cold deionized distilled water (10 mg/ml) on 100 × 1.6 cm column of P10 Bio-Gel (Biorad, UK) to purify the FML. The collected FML samples were analyzed further for the carbohydrate content and for the presence of 10–96 kDa bands corresponding to FML glycoprotein on 10% SDS-PAGE. The purified FML samples were lyophilized and stored at -20 °C for further use.

Isolation of peritoneal macrophages

Seven female BALB/c mice were sacrificed by CO₂ euthanasia. Isolation of peritoneal macrophages was performed based on the procedure described by Bibak et al (26). Brieﬂy, murine peritoneal cells were harvested by lavage of the peritoneal cavity with 10 ml of RPMI 1640 medium (Invitrogen, Germany). The cells were centrifuged at 200×g for 10 min and washed them in PBS/cold ddH₂O. The cells were then cultured in RPMI 1640 medium in petri dishes at 37 °C for 4 h. In this experiment, we considered three control groups: 1. PBS group: activated macrophages were cultured in the presence of PBS alone at the same volume as the other additions (10μl/ml), 2. GL group: activated macrophages treated with GL (5 μg/ml) 3. FML group: activated macrophages treated with FML (5 μg/ml). Petri dishes were carefully washed using Hanks' solution to remove the non-adherent cells. The cells adhered to the petri dishes were trypsinized and the concentration of the cells were adjusted to 1 × 10⁶ cells/ml in the RPMI medium (RPMI medium with 10% FCS (Invitrogen, Germany) containing 50 IU/ml penicillin/streptomycin (Sigma-Aldrich, USA).

Treatment of peritoneal macrophages with a combination of FML and GL

Isolated peritoneal macrophages were stimulated with 10 μg/ml of LPS at 37 °C and 5% CO₂ for 4 hr. The activated macrophages were treated with FML together with varying concentrations of GL to assess the immunomodulatory eﬀects of combination of FML and GL. To prepare activated macrophages, 2 × 10⁵ macrophage cell suspensions (200 μl/well on 96-well ﬂat-bottom plates) in each well, were treated with 10 μg/ml of LPS from Escherichia coli O111: B4 (Sigma, USA) in a complete RPMI medium. Thereafter, 5 μg/ml of FML and 1, 10, or 20 μg/ml of GL (Sigma, USA) mixtures were added to the activate cells in the wells in triplicate as described earlier (25, 27). The cells were then cultured at 37 °C and 5% CO₂ for 48hr. In the control group or PBS group, activated macrophages were cultured in the presence of PBS alone at the same volume as the other additions (10μl/ml). For NO assay, we used only complete RPMI-1640 medium as the blank control group. After 48h, culture supernatants from each well of 96-well plate were collected and stored at -80 °C until further analysis. Each experiment was performed in triplicate. The different study groups are shown in Table 1. MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (Merck, Germany) assay was performed to evaluate the macrophage viability after 48hr of incubation at 37 °C with different concentrations of GL (0.1, 1, 10, 20, 50 and 100 μg/ml).

Table 1: Treatment of peritoneal macrophages with GL, FML and combination of FML and different concentrations of GL in presence of LPS.

No. Study group	1	2	3	4	5	6
Treatment	LPS (10 μg/ml) + PBS^*	LPS (10 μg/ml) + GL (5 μg/ml)	LPS (10 μg/ml) + FML (5 μg/ml)	LPS (10 μg/ml) + FML (5 μg/ml) + GL (1 μg/ml)	LPS (10 μg/ml) + FML (5 μg/ml) + GL (10 μg/ml)	LPS (10 μg/ml) + FML (5 μg/ml) + GL (20 μg/ml)

* Phosphate buffered saline.

Nitric oxide and cytokine assay

The culture supernatants were evaluated for the stable end-products of NO, nitrates, and nitrites, using the Standard Griess Reagent according to the instructions in the manual (Cayman Chemical, USA). Levels of NO in different treatment groups were determined by reading the absorbances at 540 nm in a microplate reader (BioTek). The mean optical density (OD) values of the blank were subtracted from the mean OD values of the test groups. The concentration of the nitrite was calculated from the standard curve obtained with serial dilutions of sodium nitrite as the standard. Presence of TNF-α, IL-10 and IL-12p70 were measured in the cell culture supernatants using sandwich ELISA kits according to the instructions of the manufacturer (eBioscience, USA). The minimum detectable concentration was 5 pg/ml for both IL-12p70 and IL-10, and 1 pg/ml for TNF-α.

Statistical analysis

GraphPad Prism software version 5.0 (GraphPad Software, USA) was used for statistical analysis of the data. Data distribution was analyzed by Kolmogorov–Smirnov test. According to the results of the normality test, one-way ANOVA followed by Dunns or Tukey post-test or non-parametric Kruskal–Wallis test were used for statistical comparisons. Data are shown as the mean ± SD of three independent experiments. P values < 0.05 were considered statistically significant.

After the treatment of LPS stimulated macrophages with FML and different concentrations of GL, we determined the viability of cells with MTT reduction assay. Similarly, measured the levels of TNF-α, IL-10 and IL-12p70 using sandwich ELISA method and NO concentration using Griess reaction.

Results of the MTT assay indicated that co-treatment of macrophages with FML (5μg/ml) and GL (1, 10, and 20 μg/ml) had no cytotoxic effect on the activated macrophages, thus these concentrations were used for NO and cytokine assay. Since the activated macrophages treated with GL at 50 and 100 μg/ml exhibited low viability (<90%) and hence were not included for further analysis (Fig. 1).

Fig. 1: Peritoneal macrophage viability in the presence of different concentrations of Glycyrrhizin (GL). The average values of MTT reduction in activated macrophages treated with different concentrations of GL were the same in all the groups except at concentrations of 50 and 100 μg/ml (P > 0.05).

The concentrations of NO in the supernatants of the activated macrophages treated with FML were significantly higher than the activated macrophages treated with PBS (11.20 ± 1.80 μM/ml vs. 7.84 ± 1.54 μM/ml, ^*P=0.013). As shown in Fig. 2, treatment of activated macrophages with FML (5 μg/ml) plus GL (at concentration of 10 and 20 μg/ml) significantly increased the NO production in comparison with FML treatment alone (11.20 ± 1.80 μM/ml vs. 14.40 ± 1.90 μM/ml, ^*P= 0.026 and 15.60 ± 1.66 μM/ml, ^**P= 0.003) (Fig.2).

Fig. 2. The effect of the combination of Fucose-Mannose Ligand (FML) with Glycyrrhizin (GL) on the production of Nitric Oxide (NO) by activated macrophages. Co-treatment with FML and GL significantly increased NO production in activated macrophages in comparison with activated macrophages treated with PBS, FML or GL alone. Results represent mean (mean ±SD) of three independent experiments with macrophages from seven mice per experiment (*P < 0.05, **P < 0.01, and ***P < 0.001). PBS: Phosphate-buffered saline.

The measurement of TNF-α in the culture supernatant of activated macrophages, showed no significant differences between the FML-treated macrophages and PBS treated macrophages (577.6±73.4 pg/ml vs. 526±45.6 pg/ml, P= 0.223). However, the results of our study that were treated with FML in combination with 20 μg/ml GL showed significant increase in the production of TNF-α in activated macrophages compared to the activated macrophages treated with FML alone (782.0 ± 69.49 vs. 577.6±73.4 pg / ml, ^**P=0.0019) (Fig. 3).

Fig. 3. The effect of the treatment with combination of Fucose-Mannose Ligand (FML) with Glycyrrhizin (GL) on the production of TNF-α by activated macrophages. The production of TNF-α was significantly higher in the macrophages treated with the combination of FML and GL at high concentration (20 μg/ml) compared to the activated macrophages treated with PBS, FML or GL alone (^**P=0.0019). Results represent mean (Mean ± SD) of the three independent experiments with macrophages from seven mice per experiment (*P < 0.05, **P < 0.01, and ***P < 0.001). PBS: Phosphate-buffered saline.

The FML significantly increased IL-10 production from activated macrophages compared to the activated macrophages treated with PBS (1601 ± 54.11 pg/ml vs. 1242 ± 79.68 pg/ml, ^**P=0.005). The concentration of IL-10 in the supernatant of the activated macrophages treated with combination of FML and GL (at concentrations of 10 and 20 μg/ml) were significantly lower than the concentration of IL-10 in the supernatant of only FML-treated activated macrophages (1601 ± 54.11 pg/ml vs. 1028±46.2 pg/ml, ^***P<0.001, and 722.2±147.8 pg/ml, ^***P<0.001) (Fig. 4).

Fig. 4. The effect of the combination of Fucose-Mannose Ligand (FML) with Glycyrrhizin (GL) on the production of IL-10 by activated macrophages. Treatment with FML together with GL at 10 and 20 μg/ml concentrations significantly reduced IL-10 production by activated macrophages compared to the FML treatment alone. Results are expressed as mean ± SD of triplicate cultures. Results represent mean (mean ± SD) of three independent experiments with macrophages from seven mice per experiment (*P < 0.05, **P < 0.01, and ***P < 0.001). PBS: Phosphate-buffered saline.

The results of our study showed that the co-treatment with FML and GL (at concentrations of 10 and 20 μg/ml) significantly increase the production of IL-12p70 from activated macrophages compared with the activated macrophages treated with FML alone (749.3 ± 47.5 pg/ml vs. 991.6± 79.1 pg/ml, **P < 0.001, and 964.6± 83 pg/ml, ***P < 0.0004 ) (Fig.5). Also, there was a significant difference between concentration of IL-12p70 produced from the activated macrophages treated with FML and the activated macrophages treated with PBS (749.3 ± 47.5 pg/ml vs. 514.6 ± 48.37 ^**P=0.002) (Fig.5).

Fig. 5. The effect of combination of Fucose-Mannose Ligand (FML) with Glycyrrhizin (GL) on the production of IL-12p70 by macrophages. The treatment with FML and with 10 and 20 μg/ml concentrations of GL significantly increased IL-12p70 levels in the activated macrophages. Results are expressed as mean ± SD of measurements from triplicate cultures. (*P < 0.05, **P < 0.01, and ***P < 0.001). PBS: Phosphate-buffered saline.

Previous studies have shown that FML does not provide adequate immunogenicity and cannot efficiently stimulate immune response of the macrophages (9, 15). GL is a well-known immunomodulatory component that stimulates immune response of the infected macrophages (28). Therefore, we assumed that treatment with a combination of FML and GL can improve the efficiency of macrophages against VL infection through the induction of protective cytokines and reactive nitrogen species (RNS). To evaluate this hypothesis, we studied the effects of FML in combination with GL on the production of TNF-α, IL-12p70, IL-10 and NO in the murine peritoneal activated macrophages in vitro.

The results of our study indicated that the treatment of activated macrophages with FML plus GL leads to enhanced production of NO, TNF-α, IL-12p70 in comparison with FML treatment alone. Surprisingly, we found that co-treatment of macrophages with FML and GL markedly inhibits the production of IL-10 compared to FML treatment alone. Cytokine profile and NO levels in macrophages treated with a combination of FML and GL were similar to the patterns described in earlier studies on the M1 macrophages (16).

NO has been demonstrated to be a principal effector molecule responsible for mediating the intracellular killing of Leishmania parasites, particularly L. donovani complex (29). The current study has shown that GL helps in enhancing the production of NO from FML-treated activated macrophages (Fig.2). In accordance with the present results, previous studies have demonstrated that FML or GL alone enables increase of NO production from activated macrophages (19, 24).

The TNF-α plays a critical role in the control of intracellular pathogens, especially those that infect macrophages (30). It has been demonstrated that TNF-α is required for the control of VL infection in human through the stimulation of IFN-γ production (30). Similarly, Tumang et al. have also shown that endogenous TNF-alpha appears to be critical for both the initial acquisition of resistance to L. donovani and resolution of experimental VL infection (31). We know from our previous study that FML is unable to significantly enhance the TNF-α levels in the activated macrophages (19). In the current study, we found that the treatment of activated macrophages with FML plus GL significantly increases the production of TNF-α, while treatment with FML or GL alone did not increase the production of TNF-α significantly. This outcome is contrary to that of Liu et al. and Fu et al. who found that GL inhibits the secretion of TNF-α by LPS-stimulated macrophages and mammary epithelial cells, respectively (32, 33). It is difficult to explain these discrepancies, but it may be due to inhibition of LPS-induced nuclear factor-ĸB by GL without suppression of nuclear factor-ĸB induced by MyD88-dependent downstream signaling (33).

VL is characterized by the absence of cytokines such as IFN-γ and IL-12 and cure of VL is associated with restoration of these cytokines (34). Several reports have shown that IL-10 and IL-12 play opposite roles in control of VL infections (35). The IL-10 is associated with progression VL and IL-12 with the control of disease caused by Leishmania donovani complex (35). In a recent study, we demonstrated that FML increases the levels of both IL-12p70 and IL-10 in activated macrophages (19). In the present study, we have shown that use of FML in combination with GL exerts strong booster effect on IL-12 and suppressive effect on IL-10 production in activated macrophages. This finding is consistent with that of Dai et al. who demonstrated the ability of GL to enhance LPS-induced IL-12 production by peritoneal macrophages (23). The results of the present study are also consistent with our earlier observations, which showed that GL significantly inhibits IL-10 secretion by LPS-stimulated RAW264.7 cells, a mouse macrophage cell line (32).

Taken together, our findings suggest that GL can improve immunostimulatory effect of FML on macrophages and leading to their polarization toward an M1-like phenotype, which is an efficient phenotype against Leishmania parasites. More studies using animal models of VL are needed to evaluate the protective effect of the combinatorial treatment with FML/GL.

Visceral leishmaniasis (VL), Fucose-Mannose Ligand (FML), Lipopolysaccharide (LPS), Nitric Oxide (NO), Glycyrrhizin (GL), Phosphate buffered saline (PBS), Microwave Theory and Techniques (MTT), Optical Density (OD)

Ethics approval and consent to participate

The experimental protocols that have been used in this research was approved by the Ethical Committee of the North Khorasan University of Medical Sciences, Bojnurd, Iran (IR.NKUMS. REC.1395.06).

Consent for publication

Not applicable

Availability of data and materials

Data supporting the conclusions of this article are included within the article.

Competing interests

Not applicable

Funding

This work was supported by North Khorasan University of Medical Sciences, Bojnurd, Iran (grant No. 95/P/914).

Authors' contributions

Dr. Namdar and Dr. Shafiei performed the experiments and prepared the draft of the manuscript, Dr. Hatam helped with the preparation of FML and Ag. Dr. Zolfaghari Emameh and Dr. Aspatwar have helped with the scientific design of the study and writing of the manuscript.

Disclosure statement

The authors reported no potential conflict of interest relevant to this article.

Acknowledgments
The authors thank the members of the Department of Pathobiology and Medical Laboratory Sciences, North Khorasan University of Medical Sciences, Bojnurd, Iran for their technical assistance.

Mohebali M, Moradi-Asl E, Rassi Y. Geographic distribution and spatial analysis of Leishmania infantum infection in domestic and wild animal reservoir hosts of zoonotic visceral leishmaniasis in Iran: A systematic review. J Vector Borne Dis. 2018;55:173-83.
Mirahmadi H, Mansouri Nia M, Ebrahimzadeh A, Mehravaran A, Shafiei R, Rahimi MT, Zolfaghari Emameh R, Barker HR. Genotyping determination of Acanthamoeba strains: an original study and a systematic review in Iran. J Water Health. 2019;17:717-727.
Shafiei R, Ghatee MA, Jafarzadeh F, Javanshir Z, Karamian M. Genotyping and phylogenetic analysis of unusually located hydatid cysts isolated from humans in north-east Iran. J helminthol.2019;94:e64.
Sarkari B, Parhoode M, Khabisi SA, Shafiei R, Mohammadi-ghalehbin B. Genetic diversity of Fasciola spp. isolates from northern part of Iran: comparison with southwestern isolates. J Parasit Dis.2017;41:768-772.
Sarkari B, Lari M, Shafiei R, Sadjjadi SM. A comparative seroprevalence study of toxocariasis in hypereosinophilic and apparently healthy individuals. Arch Pediatr Infect Dis.2015;3:e17911.
Ponte-Sucre A, Gamarro F, Dujardin J-C, Barrett MP, López-Vélez R, García-Hernández R, et al. Drug resistance and treatment failure in leishmaniasis: A 21st century challenge. PLoS neglected tropical diseases. 2017;11:e0006052.
Moafi M, Rezvan H, Sherkat R, Taleban R. Leishmania vaccines entered in clinical trials: A review of literature. Int J Prev Med. 2019;10:95.
Chakravarty J KS, Trivedi S, Rai VK, Singh A, Ashman JA, Laughlin EM, et al. A clinical trial to evaluate the safety and immunogenicity of the LEISH-F1+MPL-SE vaccine for use in the prevention of visceral leishmaniasis. Vaccine. 2011;29:3531-7.
Jain K, Jain NK. Vaccines for visceral leishmaniasis: A review. J Immunol Methods. 2015;422:1-12.
Duarte MC, Lage DP, Martins VT, Chavez-Fumagalli MA, Roatt BM, Menezes-Souza D, et al. Recent updates and perspectives on approaches for the development of vaccines against visceral leishmaniasis. Rev Soc Bras Med Trop. 2016;49:398-407.
Evans KJ, Kedzierski L. Development of vaccines against visceral leishmaniasis. J Trop Med. 2012;2012:892817.
Zolfaghari Emameh R, Barker H, Hytonen VP, Tolvanen ME, Parkkila S. Beta carbonic anhydrases: novel targets for pesticides and anti-parasitic agents in agriculture and livestock husbandry. Parasit Vectors. 2014;7:403.
Das A, Ali N. Vaccine development against Leishmania donovani. Front Immunol. 2012;3: 99.
Saraiva EM dFBA, Santos FN, Borja-Cabrera GP, Nico D, Souza LO, de Oliveira Mendes-Aguiar C, de Souza EP, Fampa P, Parra LE, Menz I, Dias Jr JG, de Oliveira SM, Palatnik-de-Sousa CB. The FML vaccine (Leishmune) against canine visceral leishmaniasis: a transmission blocking vaccine. Vaccine. 2006;24:2423–31.
Palatnik-de-Sousa C, Dutra H, Borojevic R. Leishmania donovani surface glycoconjugate GP36 is the major immunogen component of the Fucose-Mannose Ligand (FML). Acta tropica. 1993;53:59-72.
Tomiotto-Pellissier F, da Silva Bortoleti BT, Assolini JP, Gonçalves MD, Carloto ACM, Miranda-Sapla MM, et al. Macrophage polarization in Leishmaniasis: Broadening Horizons. Frontiers in immunology. 2018; 9: 2529.
Faraji F, Karjoo Z, Moghaddam MV, Heidari S, Emameh RZ, Falak R. Challenges related to the immunogenicity of parenteral recombinant proteins: Underlying mechanisms and new approaches to overcome it. International reviews of immunology. 2018;37:301-15.
Shafiei R, Namdar Ahmadabad H, Nezafat Ferizi M, Bakhshi Joibari F, Ghahramani A, Hatam GR, et al. Cytokine profile and nitric oxide levels in macrophages exposed to Leishmania infantum FML. Exp Parasitol. 2019;203:1-7.
Bordbar N, Karimi MH, Amirghofran Z. The effect of Glycyrrhizin on maturation and T cell stimulating activity of dendritic cells. Cell Immunol. 2012;280:44-9.
Yamashita T, Asano Y, Taniguchi T, Nakamura K, Saigusa R, Miura S, et al. Glycyrrhizin ameliorates fibrosis, vasculopathy, and inflammation in animal models of systemic sclerosis. J Invest Dermatol. 2017;137:631-40.
Yoshida S, Lee JO, Nakamura K, Suzuki S, Hendon DN, Kobayashi M, et al. Effect of glycyrrhizin on pseudomonal skin infections in human-mouse chimeras. PLoS One. 2014;9:e83747.
Deng Q-P, Wang M-J, Zeng X, Chen GG, Huang R-Y. Effects of glycyrrhizin in a mouse model of lung adenocarcinoma. Cell Physiol Biochem. 2017;41:1383-92.
Dai JH, Iwatani Y, Ishida T, Terunuma H, Kasai H, Iwakula Y, et al. Glycyrrhizin enhances interleukin‐12 production in peritoneal macrophages. Immunology. 2001;103(2):235-43.
Yi H, Nakashima I, Isobe K-i. Enhancement of nitric oxide production from activated macrophages by glycyrrhizin. Am J Chin Med. 1996;24:271-8.
Foroughi-Parvar F, Hatam GR, Sarkari B, Kamali-Sarvestani E. Leishmania infantum FML pulsed-dendritic cells induce a protective immune response in murine visceral leishmaniasis. Immunotherapy. 2015;7:3-12.
Bibak B, Gharib FG, Daneshmandi S, Abbaspour AR, Firizi MN, Ahmadabad HN. The immunomodulatory effects of abortion-prone mice decidual and serum soluble factors on macrophages and splenocytes. Eur J Obstet Gynecol Reprod Biol. 2012;165:331-6.
de Lima VM, Ikeda FA, Rossi CN, Feitosa MM, Vasconcelos RO, Nunes CM, et al. Diminished CD4+/CD25+ T cell and increased IFN-gamma levels occur in dogs vaccinated with Leishmune in an endemic area for visceral leishmaniasis. Vet Immunol Immunopathol. 201015;135:296-302.
Mao Y, Wang B, Xu X, Du W, Li W, Wang Y. Glycyrrhizic acid promotes M1 macrophage polarization in murine bone marrow-derived macrophages associated with the activation of JNK and NF-κB. Mediators Inflamm. 2015;2015:372931.
Sarkar A, Saha P, Mandal G, Mukhopadhyay D, Roy S, Singh SK, et al. Monitoring of intracellular nitric oxide in leishmaniasis: its applicability in patients with visceral leishmaniasis. Cytometry A. 2011;79:35-45.
Singh N, Kumar R, Nylén S, Sacks D, Sundar S. The effect of TNF-α neutralization on parasite load and cytokine production in human visceral leishmaniasis. Int J Infect Dis. 2016;45:62.
Tumang MC, Keogh C, Moldawer LL, Helfgott DC, Teitelbaum R, Hariprashad J, et al. Role and effect of TNF-alpha in experimental visceral leishmaniasis. J Immunol. 1994;153:768-75.
Liu Z, Zhong JY, Gao EN, Yang H. Effects of glycyrrhizin acid and licorice flavonoids on LPS-induced cytokines expression in macrophage. Zhongguo Zhong Yao Za Zhi. 2014;39:3841-5.
Fu Y, Zhou E, Wei Z, Liang D, Wang W, Wang T, et al. Glycyrrhizin inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model. The FEBS journal. 2014;281:2543-57.
Bacellar O, D'Oliveira A, Jr., Jeronimo S, Carvalho EM. IL-10 and IL-12 are the main regulatory cytokines in visceral leishmaniasis. Cytokine. 2000;12:1228-31.
Dayakar A, Chandrasekaran S, Kuchipudi SV, Kalangi SK. Cytokines: Key determinants of resistance or disease progression in visceral leishmaniasis: Opportunities for novel diagnostics and immunotherapy. Front Immunol. 2019;10:670.

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Cytokine profile and nitric oxide levels in peritoneal macrophages of BALB/c mice exposed to Fucose-Mannose Ligand of Leishmania infantum combined with Glycyrrhizin

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