Abstract
The immune response of the intermediate host with alveolar echinococcosis was investigated on mice intraperitoneally infected with Echinococcus multilocularis protoscoleces. The study was focused on cell-mediated immune response (dependent on interactions of T lymphocytes and macrophages), which is considered protective in alveolar echinococcosis. The immune response to E. multilocularis is regulated by Th1/Th2 cytokines produced by the CD4+ T lymphocyte subpopulation. Metacestode has been known for its ability to modify immune functions and suppress effective specific cell response to ensure its survival in host organism. The influence of immunomodulatory substances – muramyltripeptide (L-MTP-PE), glucan (GI), glucan with zinc (GIZn), and transfer factor (TF) – applied alone or combined with anthelmintic albendazole (ABZ) on regulative and effector components of immunity were tested and at the same time, antiparasitic efficacy of immunomodulators was evaluated.
Keywords
- Echinococcus multilocularis
- therapy
- muramyltripeptide
- glucan
- transfer factor
1. Introduction
The larval stage of
In our study, we focused on the activity of three immunomodulators (liposomized muramyltripeptide, glucan with zinc, and transfer factor) in enhancing of the host antiparasite defence and the efficacy of anthelmintic albendazole treatment in alveolar echinococcosis.
2. Materials and methods
Experiments were performed on pathogen-free BALB/c mice, males, weighing 20–25 g. Mice were kept under a 12-h light/dark regime at room temperature (21±3°C) and 50–60% relative humidity on a commercial diet and water. The experimental protocols complied with the current Slovak ethics law.
2.1. Infection
Parasite cysts were isolated 4 months post infection (p.i.) and cut into pieces in sterile RPMI 1640 medium (Sigma-Aldrich, Germany) supplemented with antibiotics, 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, Germany), and passed through a Cell Dissociation Sieve Tissue Grinder Kit using apertures ranging from 380 to 45,7 μm (Sigma-Aldrich, Germany).
Protoscoleces obtained after the last filtration were maintained in RPMI and counted for an infective dose.
2.2. Efficacy of treatment
The antiparasitic efficacy of immunotherapy was evaluated by the cyst development in infected mice.
2.3. T and B lymphocyte proliferation assay
The spleen was aseptically homogenized in phosphate-buffered saline (PBS) (pH 7.2) to obtain cells. Cell suspension was washed twice with PBS and finally with RPMI 1640 medium (Sigma-Aldrich, Germany). Erythrocytes were removed by lysis in hypotonic solution (0.85 % NH4Cl) and lymphocytes were resuspended to a final concentration of 5 x 106 cells /ml in RPMI 1640 medium. The assay was performed in 96 wells plates (Nunc, Denmark) and cells were incubated in RPMI 1640 medium (100 µl) containing 10 % bovine fetal serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Mitogens Concanavalin A (Con A) (T cells) and lipopolysaccharide (LPS) (B cells) (Sigma-Aldrich, Germany) were added in a dose 100 µl (concentration 10 µg/ml) to the cell suspensions and incubated at 37 °C in 5 % CO2 and 85 % humidity for 72 h. Then 20 µl of 3,4-dimethylthiazolyl 2,5-diphenyltetrazolium bromide (Sigma-Aldrich, Germany) (0.1 % solution) was added to the cell suspensions and incubated at 37 °C and 5 % CO2 for 4 h followed by centrifugation at 800 x
SI= E540 – E630 (stimulated cells) / E540 – E630 (unstimulated cells)
Proliferative responses were measured separately for lymphocytes isolated from each mouse per group.
2.4. Number of CD4+ and CD8+ T cells
Lymphocytes from the spleens and depleted of erythrocytes were resuspended in PBS (pH 7.2) at a final concentration of 1x 106 cells /ml. Monoclonal antibodies rat anti-mouse CD4 fluorescein isothiocyanate-conjugated and rat anti-mouse CD8 phycoerythrin-conjugated monoclonal antibodies (BD Biosciences PharMingen, Belgium) were used at the concentration of 0.4 µg/106 cells at 4°C for 30 min. After washing in PBS three times, cells were analyzed by the FACScan flow cytometer (Becton Dickinson Biosciences, Germany) and CellQuest software. Cells from each mouse per group were analyzed individually. The final numbers of both cell populations were calculated as proportion from the total isolated lymphocytes per spleen/mouse.
2.5. Concentration of IFN-γ and IL-5 in serum
The capture ELISA was employed to determine the concentration of cytokines IFN-γ and IL-5 in serum according to the method [22]. IFN-γ and IL-5 were used as marker cytokines for the Th1 and Th2 responses, respectively. Cytokine-specific monoclonal antibodies were used, for IFN-γ detection: pure anti-mouse IFN-γ (R4-6A2) and biotin anti-mouse IFN-γ (XMG1.2); for IL-5 detection: pure anti-mouse IL-5 (TRFK5) and biotin anti-mouse IL-5 (TRF4) (all BD Biosciences PharMingen, Belgium). Results were expressed at pg/ml using murine recombinant IFN-γ and IL-5 (BD Biosciences PharMingen, Belgium) as standards. The detection limit of the assay for the both cytokines was 40 pg/ml.
2.6. Superoxide anion assay
Production of superoxide anion (O2-) by peritoneal macrophages was detected as superoxide dismutase (SOD) – reduction of ferricytochrome C with and without stimulation with phorbol myristate acetate (PMA) [23]. Cells were obtained by peritoneal lavage and after washing in PBS were diluted at concentration of 1 x 106 cells/ml in RPMI 1640 (Sigma-Aldrich, Germany). Cell suspension (1 ml/well) was added to 24-well plate (Falcon, France) and incubated at 37 oC in 5 % CO2 and 85 % humidity for 2 h. Nonadherent cells were removed by washing with ice-cold Earls Balanced Salt Solution (EBSS) (pH 7.2). The reaction was carried out in 0.3 ml/well of 160 µM ferricytochrome C (Sigma-Aldrich, Germany) in EBSS. In control, the reaction was immediately blocked by 300 µg SOD/10 µl in EBSS. The stimulation of cells was induced by 10 µl of PMA in ethanol. Cells were incubated at 37 oC in 5 % CO2 and 85 % humidity for 2 h. Supernatant from wells was centrifuged at 170 x
2.7. Statistical evaluation
Statistical differences were assessed using Kruskal-Wallis ANOVA and post hoc Tukey’s HSD test (a value of p<0.05 was considered significant) in the program Statistica 6.0 (Stat Soft, Tulsa, USA) statistical package.
3. Muramyltripeptide
Muramylpeptides – components of bacterial cellular wall are classified as biological immunomodulators. Muramylpeptides primarily activate macrophages to a high production of oxygen radicals and a secretion of inflammatory cytokines, which activate neutrophils, T and B lymphocytes [24]. Muramyldipeptide (MDP) is the smallest bacterial structure with immunopotent activity. The positive effect of MDP on the host immune response was also observed in several parasitic infections [25–27]. Muramyltripeptide phosphatidylethanolamine (MTP-PE) is a lipofilic derivate of MDP. Liposome-encapsulated MTP-PE has an enhanced effect on macrophages to secrete pro-inflammatory cytokines, which results in increasing of cytotoxicity of these cells [28–30].
Affinity of liposomized forms of MTP-PE to the reticuloendotelial system, in particular to macrophages located in the liver and the spleen [30, 31], could be useful in therapy of alveolar echinococcosis, by which the parasite cysts primarily develop in the liver. The biological effect of MTP-PE takes place in Kuppfer cells in the liver (macrophage’s equivalent) [32]. Macrophages activated by liposomized MTP-PE stimulate Th1 subpopulation of lymphocytes and proinflammatory mediators via cytokine secretion [28, 29, 33, 34]. T lymphocytes therefore constitute an active component of immune reactions after immunomodulation with muramylpeptides.
The effect of muramyltripeptide phosphatidylethanolamine incorporated into multilamellar liposomes (L-MTP-PE) on immune response of intermediate host infected with
3.1. Experimental design
Experiments were carried out on male BALB/c mice (n=150) weighing 20–25 g. Mice were randomly divided into five groups as follows:
Group 1 – uninfected and untreated (control)
Group 2 – infected intraperitoneally with 5000
Group 3 –
Group 4 –
Group 5 –
Samples of blood, spleen, and peritoneal macrophages were obtained at the following weeks: 0 (prior infection), 2, 4, 8, 10, 12, 14, 18, 22, and 26 p.i. from all groups (3 mice per experimental day).
4. Results and discussion
In our experiment, the
However, the application of L-MTP-PE to infected mice had a positive stimulatory effect on T and B lymphocytes, especially in combination of L-MTP-PE+ABZ, where proliferative activity of lymphocytes was increased for a long time, from week 8 p.i. (2 weeks after the end of the therapy) till week 14 p.i. (lasted almost 2 months). Muramylpeptides belongs to the polyclonal activators of B lymphocytes, inducing a high proliferation and production of polyclonal antibodies. Muramyldipeptide activates B cells particularly during a late phase of their differentiation because the specific receptors for MDP are expressed on B cell surface at a definitive stage of their maturation [35].
T lymphocytes play a major role in the control of immune response in intermediate host organism infected with
Previous experimental studies have manifested that
In many parasitic infections, the clinical outcome of the disease is associated with Th1 or Th2 cell activation.
Liposomized MTP-PE suppressed the Th2 response, associated with a progressive development of larval cysts in
Macrophages participate in the destruction of the parasite through synthesis of O2- and other free radicals and, together with T cells, control the parasite development [39]. In our experiment (Figure 10), therapy L-MTP-PE stimulated O2- generation in infected mice from weeks 8 to 12 p.i. (i.e. for 1 month). The combination of L-MTP-PE+ABZ significantly increased the superoxide production from weeks 8 to 18 p.i. (almost for 3 months).
It documents a long-term and strong activation of macrophage’s metabolism accompanied with a huge release of other biologically effective substances. In parallel, the proliferative activity of B cells was also increased, which could be related to macrophages’ stimulation induced by B cell secrets [46]. Positive effect of L-MTP-PE on macrophages had been confirmed in immunosuppressed mice [25], in which the immunomodulator induced a macrophages restoration. An induction of cytotoxicity of liver macrophages against tumor cells after stimulation with L-MTP-PE was observed [28]. In addition, an increased tumoricidal activity of Kupffer cells in mice after application of L-MTP-PE was recorded [47]. Their results were later corroborated in the work [48], which described an activation of Kupffer cells with an increased production of superoxide anion and subsequent reduction of micrometastases in rats after MTP-PE therapy.
Macrophages’ stimulation
Potentiation of effector components of the immunity after the immunomodulation and anthelmintic therapy of
In conclusion, our results show the possibility to increase antiparasitic efficacy of ABZ in
5. Glucan and zinc
Glucans are β-(1,3)-D polymers of glucose occuring naturally as the basic component of the cell walls of bacteria, fungi, and yeast [52–54]. Glucans activate mainly nonspecific stimulation of the immune system [22], particularly proliferation and functional activity of phagocytic cells (macrophages, NK cells) as the part of innate immunity [55, 56], but also have an influence on specific immunity – increase of the T-lymphocyte activity and production of circulating antibodies [57–59]. The immunostimulative effect of glucan could be increased by its combination with the other components – immunoglobulin G, zinc, or vitamin C [60, 61]. Zinc interacts with cytokines and proteases and indirectly influences the immune system [62]. Its immunostimulative effect on T lymphocytes, macrophages has been known [63–65] and so zinc could potentiate the immunostimulative effect of the glucan during therapy of alveolar echinococcosis. Also living
The role of glucan immunomodulator (GI, soluble β-(1,3)-D glucan) and/or glucan immunomodulator supplemented with zinc (GIZn) in stimulation of a host defence mechanism against
5.1. Experimental design
Experiments were carried out on male BALB/c mice (n=220) weighing 20–25 g. Mice were divided randomly into five groups as folows:
Group 1 – uninfected and untreated (control)
Group 2 – infected intraperitoneally with 5000
Group 3 –
Group 4 –
Group 5 –
Samples of blood, spleen, and peritoneal macrophages were obtained at the following weeks: 0 (prior infection), 2, 4, 6, 8, 10, 12, 14, 18, 22, and 26 p.i. from all groups (four mice per experimental day).
6. Results and discussion
The
Immunomodulators GI and GIZn did not induce such stimulative effect on B cell proliferation (Figure 13); they restored a suppressed proliferative activity of B cells only for a short time. The similar restoring effect of glucan was observed in other parasitic infections with
CD4 T cells represent the main T subpopulation in the first phase of alveolar echinococcosis in granuloma formation around the parasite [17, 79]. In our experiment (Figure 14), the presence of CD4 T cells was stimulated from weeks 6 to 8 p.i. in the spleen of mice infected and treated with GI+ABZ. Supplementation of glucan with zinc could contributed to prolong an increase in the CD4 T cell subpopulation in mice treated with GIZn+ABZ from week 6 to 14 p.i. It has been known that zinc deficiency reduces the production of IL-4 cytokine acting as a growth factor for helper CD4 T cells [80]. The long-term stimulation of CD4 T cells after GIZn+ABZ therapy could increase antiparasite defence. Periparasitic granuloma in regressive alveolar echinococosis is composed of great numbers of CD4 T lymphocytes, macrophages, and myofibroblasts [81]. In patients with progressive disease, the CD8 T lymphocytes are dominant [82]. In our experiment,
The Th1 and Th2 cytokine balance regulates the immune response to
IFN-γ is widely recognized as a major priming signal for macrophages’ activation, which have a key role in effector phase of immune response to
A notable finding in the treatment efficacy was that GI’s presence in GI+ABZ therapy partly inhibited the parasitostatic effect of ABZ (Figure 18).
It could be explained by GI’s stimulatory effect on fibrosis in periparasitic granuloma, which could inhibit anthelmintic drug from penetrating to parasite vesicle. The glucan’s profibrotic activity in metacestode infection
In our experiment, the stimulation of IFN-γ production induced by GIZn could reduce the irreversible fibrosis in periparasitic granuloma and thus allow penetration of anthelmintics to larvocyst. IFN-γ has not only an antifibrotic property, but it is also engaged in Th1 immune reactions, which are protective against
Zinc plays an important role as cofactor of enzymes involved in collagen synthesis [89] and zinc supplemention has favorable inhibitive effects on hepatic fibrosis [90, 91]. This fact can explain better the antiparasitic efficacy of treatment with GIZn and nearly twofold higher efficacy of GIZn+ABZ therapy in comparison with ABZ treatment (Figure 18).
According to host’s immunosuppression and zinc deficiency in patients with alveolar echinococcosis [67], the combination of immunomodulator GIZn and anthelmintic drug ABZ achieved the most effective control of the parasite infection. The parasitostatic effect of therapy GIZn+ABZ lasted for the longest time; also after the end of the therapy it reached the greatest reduction of
7. Transfer factor
The dialysable leucocyte extract, a product of the immune system, is known as transfer factor (TF). Transfer factors are low molecular weight dialysable products extracted from immune cells which transmit the ability to express delayed-type hypersensitivity and cell-mediated immunity from sensitized donors to nonimmune recipients [92]. TF increases macrophage activation and IL-1, IL-2, and IFN-γ production
Nonspecific transfer factor (TF) from the blood leukocytes of immunized swine was studied as an appropriate candidate for immunotherapy, supplementing conventional ABZ treatment of alveolar echinococcosis.
7.1. Experimental design
Experiments were carried out on male BALB/c mice (n=165) weighing 20–25 g. Mice were randomly divided into five groups as follows:
Group 1 – uninfected and untreated (control)
Group 2 – infected intraperitoneally with 5000
Group 3 –
Group 4 –
Group 5 –
Samples of blood, spleen, and peritoneal macrophages were obtained at the following weeks: 0 (prior infection), 2, 4, 6, 8, 10, 12, 14, 18, 22, and 26 p.i. from all groups (3 mice per experimental day).
8. Results and discussion
Transfer factors (TFs) are proteins that transfer specific cellular immunity from an immune donor to a nonimmune recipient and possess a number of nonspecific activities, such as the ability to increase the numbers of immunocompetent cells, to stimulate phagocytosis, to induce the production of interferons and interleukins, to stimulate hemopoiesis, etc. TF includes a lot of molecules, acting as antigens (MW cca 5 kDa), or acting as immunomodulators (less than 3.5 kDa) [97]. Transfer factors are very efficient in diseases in which cellular immunity plays an important role in protection and control of the disease, for example, viral, bacterial, and parasite infections, as well as immunodeficiencies and some types of cancer [98].
The host cellular immunity plays a principal role in the control of the
In our work, both the therapy with TF or combination of TF+ABZ abolished a suppressory impact of
T lymphocytes play a key role in the control of immune response in alveolar echinococcosis [18, 36]. In our study, the administration of TF alone or in combination of TF+ABZ caused the increase in numbers of CD4+ T cells in the spleen of infected mice up to week 14 p.i. (Figure 21). Patients with active alveolar echinococcosis showed increased numbers of CD8+ T lymphocytes [20] and also our results documented an increase in this subpopulation after
Active alveolar echinococcosis is related to Th2 response – dominance of IL-5 and IL-10 and week production of IFN-γ [20]. The superiority of Th1 response plays a key role in the host defence reactions against
The concentration of serum IFN-γ in mice infected with
IFN-γ (that was stimulated after TF therapy) is an important activator of macrophages and affects their energetic system [21]. Immunotherapy with TF or TF+ABZ activated the respiratory burst in macrophages with high generation of O2- for a long time, from weeks 8 to 18 p.i. (Figure 23). This is documented by a long-term stimulation of macrophages’ metabolic activity accompanied with a high production of also other biologically effective substances. At the same time, the proliferation of B cells and the IFN-γ level were increased. B cells release stimulation factors for macrophages after activation with antigens from destroyed
Activation of effector components of immune response after the immunotherapy of
In conclusion, the results of our study suggest that TF can have a positive effect on the host cellular immune response against the development of
9. Conclusion
Our results suggest the possible way to increase ABZ antiparasitic efficacy in
L-MTP-PE was recognized as a strong macrophage activator in mice infected with
GIZn+ABZ has been shown to be the most effective immunomodulatory and antiparasitic treatment, the greatest reduction of metacestode growth was observed as early as 2 weeks from the beginning of this therapy, lasting till the end of experiment (for 4 months). The addition of zinc to immunomodulatory glucan substance has greatly contributed to the reduction of intensive fibrosis after glucan treatment, which attenuated the antiparasitic effect of ABZ alone.
TF could correct the immune balance disturbance after the
Immunomodulatory agents can be beneficial in the treatment of alveolar echinococcosis, where the developed Th1 immune response supports the improvement of the anthelmintic action of benzimidazoles. The type and development of the immune response are generally managed by the lymphocyte network and its immunoregulatory mechanism. Immunomodulators could regulate the immune disbalance after the
Acknowledgments
This study was supported by the Slovak VEGA agency, grants No. 2/0172/13 and No. 2/0081/15.
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