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c mice infected with Leishmania infantum
Cytokine 95 (2017) 97–101
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Cytokine journal homepage: www.journals.elsevier.com/cytokine
Short communication
Homeopathic medicines cause Th1 predominance and induce spleen and megakaryocytes changes in BALB/c mice infected with Leishmania infantum Ana Paula Bacellar Cajueiro a,1, Ester Puna Goma a,2, Hilton Antônio Mata dos Santos a,2, Igor Almeida Rodrigues a,3, Helena Keiko Toma a,4, Silvana Marques Araújo b,5, Leoni Villano Bonamin c,6, Nelson Brêtas de Noronha Gomes d,7, Morgana Teixeira Lima Castelo-Branco e,8, Edilma Paraguai de Souza Dias f,9, Alexandre dos Santos Pyrrho a,2, Carla Holandino a,⇑ a
Graduation Program in Pharmaceutical Sciences, Universidade Federal do Rio de Janeiro, Brazil Department of Health Sciences, Universidade Estadual de Maringá, Brazil Graduation Program of Environmental and Experimental Pathology, Universidade Paulista, Brazil d Department of Veterinary Medicine, Universidade Federal de Lavras, Brazil e Biomedical Sciences Institute, Universidade Federal do Rio de Janeiro, Brazil f Graduation Program in Biological Sciences, Universidade Federal do Rio de Janeiro, Brazil b c
a r t i c l e
i n f o
Article history: Received 31 October 2016 Received in revised form 9 February 2017 Accepted 12 February 2017
Keywords: Leishmania infantum Biotherapy Antimonium crudum
a b s t r a c t The prevalence of Th1/Th2 response, spleen changes and megakaryocytes were investigated in BALB/c mice (n = 138) infected with Leishmania infantum, and treated with Leishmania infantum 30 (10 30) biotherapy – BioLi30. We performed controlled experiments using 8-to-12-week-old mice, infected with 5 107 L. infantum promastigotes, divided into eight groups: G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30 pre-treated), G4 (BioLi30 pre/post-treated), G5 (BioLi30 post-treated), G6 (Water 30 post-treated), G7 (Antimonium crudum 30 post-treated) and G8 (GlucantimeÒ post-treated). G3 –G7 groups were orally treated with their respective drugs diluted in filtered water (1:10), and G8 received GlucantimeÒ (0.6 mg/100 ml of PBS), intraperitoneally. Spleen fragments were submitted to double blind histopathological evaluation and the number of megakaryocytes was counted. Besides, animals’ serum
Abbreviations: Th1, T helper cell type 1; Th2, T helper cell type 2; IFN-c, Interferon gama; IL-4, Interleukin 4; IL-10, Interleukin 10; IL-12, Interleukin 12; AVL, American visceral leishmaniasis; BioLi30, Leishmania infantum 30 (10 30). ⇑ Corresponding author at: Laboratório Multidisciplinar de Ciências Farmacêuticas e Laboratório de Pesquisa e Desenvolvimento em Práticas Integrativas e Complementares, Departamento de Fármacos e Medicamentos, Faculdade de Farmácia, Bloco B, ss 11 e 34., Av. Carlos Chagas Filho 373, CEP: 21941-902, Centro de Ciências da Saúde (CCS), Cidade Universitária, Ilha do Fundão, UFRJ – Rio de Janeiro, RJ, Brazil. E-mail addresses: [email protected] (A.P.B. Cajueiro), [email protected] (E.P. Goma), [email protected] (H.A.M. dos Santos), [email protected] (I. Almeida Rodrigues), [email protected] (H.K. Toma), [email protected] (S.M. Araújo), [email protected] (L.V. Bonamin), [email protected] (N.B.N. Gomes), [email protected] (M.T.L. Castelo-Branco), [email protected] (E.P. de Souza Dias), [email protected] (A. dos Santos Pyrrho), [email protected] (C. Holandino). 1 Address: Graduation Program in Pharmaceutical Sciences, Multidisciplinary Laboratory of Pharmaceutical Sciences, Department of Drugs and Medicines, Faculty of Pharmacy, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco B-ss, salas 11 e 34, 21941-590 Rio de Janeiro, RJ, Brazil. 2 Address: Graduation Program in Pharmaceutical Sciences, Laboratory of Immuno-parasitology and Toxicological Analysis, Department of Social Hygiene and Clinical Analysis, Faculty of Pharmacy, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco A, sala 17, 21941-590, Brazil. 3 Address: Graduation Program in Pharmaceutical Sciences, Laboratory of Antimicrobials Prospection, Department of Natural Products and Food, Faculty of Pharmacy, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco A-ss, sala 14, 21941-590, Brazil. 4 Address: Graduation Program in Pharmaceutical Sciences, Laboratory of Molecular Diagnosis and Hematology, Department of Clinical and Toxicology Analysis, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco A, sala 13, 21941-590, Brazil. 5 Address: Department of Basic Health Sciences, Laboratory of Parasitology, Universidade Estadual de Maringá, Av Colombo 5790, Bloco I-90, sala 11, KM 130, 87020-900 Maringá, PR, Brazil. 6 Address: Graduation Program of Environmental and Experimental Pathology, Research Center of University Paulista, Department of General Pathology and Pathogenesis of Susceptibility, Universidade Paulista, Rua Dr. Bacelar 1212, 4° andar, 04026-002 São Paulo, SP, Brazil. 7 Address: Department of Veterinary Medicine, Animal Pathology Sector, Universidade Federal de Lavras, Campus Universitário, 37200-000 Minas Gerais, MG, Brazil. 8 Address: Laboratory of Cellular Immunology, Biomedical Science Institute, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco B, sala 19, 21941590, Brazil. 9 Address: Graduation Program in Biological Sciences, Bioinovar Laboratory: Unit of Bioproducts, Biocatalysis and Bioprocess, Paulo de Góes’ Microbiology Institute, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco I, sala 31, 21941-390, Brazil. http://dx.doi.org/10.1016/j.cyto.2017.02.010 1043-4666/Ó 2017 Elsevier Ltd. All rights reserved.
98 Megakaryocytes BALB/c Th1/Th2 response
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was measured after 49 days of infection, and cytokines (IFN-c, IL-4, IL-10, IL-12), as well as the Th1/Th2 correlation (IFN-c/IL-4 and IFN-c/IL-10), were analyzed. Spleen histological parameters were classified as: healthy appearance (G1); discreet (G3–G7), moderate (G2) and moderate to severe (G8) white pulp hyperplasia; proliferation of megakaryocytes (G2–G8), and intense disruption (G2–G8). All groups, except for G7, showed higher percentages of megakaryocytes per field ranging from 87% to 15%, when compared to healthy animals (G1). Th1 predominance in IFN-c/IL-4 ratio (comparing to G2) was detected in G4, G5, G6 and G7. Finally, pre/post (BioLi30x) and post-treatment (Antimonium crudum 30x) presented reduction of megakaryocytes/spleen changes due to immunomodulation animal process, controlling the infection process, probably by the Th1 cytokine predominance. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction American visceral leishmaniasis (AVL) is a serious public health problem [1]. It is a worldwide neglected disease caused by Leishmania (L.) infantum, an intracellular parasite of the mononuclear phagocyte system cells, traditionally treated with GlucantimeÒ [1]. Several studies have demonstrated immunomodulatory effects of homeopathic medicines on diseases caused by Leishmania sp. and other parasites [2–5]. Considering these promising homeopathic results, we prepared Biotherapics, following the homeopathic pharmaceutical procedure [6], and verified their efficacy. As control, we used Antimonium crudum 30, since this medication covers almost leishmaniasis symptoms totality and has been described in other works [5,7]. Thus, the therapeutic and immunomodulatory biotherapy potential [2,3,7] motivated the present work in which the efficacy of a new biotherapy prepared with infective Leishmania infantum promastigotes (BioLi30) was evaluated in animal models. In the ALV disease, there is a predominance of Th2 CD4+ lymphocytes that will produce specific cytokines, increasing IL-4 and IL-10 due to the decrease of IL-12 and IFN-c (Th1-type cytokines), for a negative feedback mechanism [8]. Therefore, it is common to observe the predominance of Th2type cytokines in infected animals and, in contrast, Th1 in treated animals, conferring infection resistance in the last one [8]. These changes in Th1/Th2 relation can be clearly observed in BALB/c mice, as they are an excellent model to evaluate the disease’s immunomodulatory effects [8]. Spleens of BALB/c infected by L. infantum presented hypertrophy and hyperplasia of the mononuclear phagocyte system, accompanied by white pulp organ’s lymphoid depletion [9]. In this organ, it is common to observe megakaryocytes, a platelet precursor cells [10], whose numerical increase has been considerably shown in some works [9,11], indicating hematopoietic activity accompanied by a parasite expansion, taking to serious spleen functional disruptions [10,11]. Thus, when tissue injury occurs, the organism tries to increase the production of platelets, since they will be responsible for maintaining the capillaries integrity [10]. In addition, platelets were recently recognized in the immune response activation, as elements involved in the elimination of pathogenic agents [4]. Therefore, the aim of this study is to evaluate the prevalence of Th1/Th2 response, histological spleen changes and megakaryocytes participation in immunomodulation process of BALB/c mice with AVL, treated with BioLi30. 2. Material and methods
kept under controlled temperature and lighting conditions, with light/dark cycle (12 h/22 ± 1 °C). 2.2. Test solutions and experimental groups BioLi30x was prepared from a suspension containing 5x107 infective promastigotes of L. infantum/mL (strain MHOM/ BR/1974/PP75 - CLIOC/Fiocruz/RJ) at a ratio of 1:10, following the Brazilian Homeopathic Pharmacopoeia [6]. To prepare the dilutions, we used sterile phosphate buffer saline (PBS) from 1 (10 1) to 6 (10 6) potencies, since our preliminary experiments indicated parasite lyses when dynamization process was done with water as solvent up to 3. To ensure the protozoa integrity, PBS was used up to 6. After this, 7 (10 7) to 30 (10 30) potencies were prepared with sterile distilled water. For dynamizations, the diluted solutions were submitted to one hundred mechanical succussions in Homeopathic Brazilian Machine (DeniseÒ-AUTIC). This procedure was performed because several works showed the biological effects of hydro-alcoholic dynamized solvents [2–5,7]. Besides, considering previous homeopathic uses of Antimonium crudum [5], we also evaluated its effects (Antimonium crudum 30) in comparison to the traditional medicine GlucantimeÒ (allopathic control). The GlucantimeÒ (Sanofi-Aventis, lot 019106, 300 mg/mL) was prepared at 0.6 mg/100 mL of sterile phosphate buffer saline (PBS) [12]. BALB/c mice were randomly divided into eight groups (n = 16–18): G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30 pre-treated), G4 (BioLi30 pre/post-treated), G5 (BioLi30 posttreated), G6 (Water 30 post-treated), G7 (Antimonium crudum 30 post-treated) and G8 (GlucantimeÒ post-treated). 2.3. Infection and treatment schemes Except for G1, all groups were infected with L. infantum promastigotes. Parasites were previously maintained in PBHIL medium supplemented with 10% of fetal bovine serum (FBS) at 27 °C [13]. At the end of log phase (120 h of growth), the cells were harvest, centrifuged (3500 rpm/5 min) and washed twice with PBS at pH 7.2. The mice were challenged with a suspension of 5 107 promastigotes/100 mL in sterile PBS, intraperitoneally. The animals were treated orally (G3–G7 groups) and intraperitoneally (G8). G3 and G4 were pre-treated for 21 days. At the end of this period, all groups were infected and, to ensure the disease establishment, there was a pause of 28 days prior the posttreatment beginning of G4–G8 for more 21 days [14].
2.1. Experimental design 2.4. Euthanasia and collection of biological materials Double blind controlled experiments using BALB/c mice (n = 138), with 8-to-12-week-old were performed. All animals were acquired at the Reference Center and Mice creation for Scientific Research of UFRJ after ethics committee approval (CEUA/UFRJ066/14). Water and feed were provided ad libitum and the animals
After 49 days of infection, mice were anesthetized with chlorhydrates of ketamine (100–150 mg/kg) and xylazine (10–15 mg/kg), intramuscularly. Sequentially, 800 lL of blood were collected by cardiac puncture and the serum centrifuged at
A.P.B. Cajueiro et al. / Cytokine 95 (2017) 97–101
10.000 rpm/10 min in centrifugal Eppendorf 5415-C, for cytokine analyzes. Euthanasia was performed by exposure to increasing CO2 atmosphere. Spleen fragments were removed, fixed in blade with 4% paraformaldehyde, and stained with hematoxylin-eosin to perform histopathological changes. The slides were observed by two independent veterinarian pathologists to avoid interpretation bias. All experimental procedures involving animals were approved by Ethics Committee (CEUA) of UFRJ, under the protocol number 066/14. 2.5. Spleen changes and quantification of megakaryocytes Histological spleen cuts were observed in optical microscope (Axyoplan 2, Zeiss/German). The spleen changes were sorted by score (0–5), indicating (in ascending order) the degree of white pulp hyperplasia with spleen disruption. The megakaryocytes were counted in six random fields. The average found in the groups were compared with the uninfected control (G1) and expressed in percentage.
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phage functions dependent of IFN-c, and IL-10 inhibits the production of IL-12) were measured in serum (ng/mL) in three/four pools at the 49th day of infection. Samples were analyzed in duplicate in ELISA 96-well plates (Costar 3590 Corning), using the commercial kit R&D Systems (USA), and read in Elisa Bio-Rad plate reader at 450 nm. Sequentially, the ratio averages of Th1/Th2 (IFN-c/IL-4 and IFN-c/IL-10) were determined by dividing the values obtained in Th1 cytokine by Th2 cytokines.
2.7. Statistical analysis One-way ANOVA was performed, followed by Tukey test to evaluate parametric variables (cytokines) and Kruskal-Wallis followed by Dunn test were performed to evaluate non-parametric variables (megakaryocytes). Graphpad Prism 5Ò software was used. P values < 0.05 were considered statistically significant.
3. Results 2.6. Cytokines and ratio Th1/Th2 The cytokines IFN-c (pro-inflammatory which activates macrophages in order to inhibit infection and modulates B lymphocytes activities: Th1 response), IL-12 (induces T CD4+ cell differentiation, which is responsible for Th1 cytokines expansion), IL4 and IL-10 (anti-inflammatories with Th2 response: IL-4 suppresses macro-
3.1. Spleen changes and quantification of megakaryocytes Spleen histological cuts presented: healthy appearance (G1); discreet (G3–G7), moderate (G2) and moderate to severe (G8; Fig. 1A) white pulp hyperplasia; proliferation of and megakaryocytes (G2–G8); and intense disruption (G2–G8; Fig. 1B and C).
Fig. 1. Spleen changes and quantification of megakaryocytes. (A) Spleen histological cuts, indicating moderate to severe white pulp hyperplasia (G2 and G8). (B) Spleen histological cuts indicating severe organ disruption (G2 and G8). (C) in set, indicating presence of severe quantity of megakaryocytes. (D) Percentage of megakaryocytes: calculated dividing the groups’ averages by G1 average (healthy animals). Statistical differences were revealed by non-parametric Kruskal-Wallis/Dunn, indicating that only G7 animals showed 10% of percentage reduction (p > 0.05) when compared to G1. All other groups showed higher values than those recorded in G1: G2 (87%; p < 0.001), G8 (53%; p < 0.001), G6 (39%; p < 0.001), G3 (29%; p < 0.05), G5 (23%; p > 0.05) and G4 (15%; p > 0.05). Compared to G2 (L. infantum), a statistically significant difference was found between this group and G3, G4, G5 and G7 (p < 0.001). Among treated groups, the following p-values were found: G7 with G3, G4, G5, G6 and G8 (p < 0.001); G4 with G6 and G8 (p < 0.01), and G5 with G6 and G8 (p < 0.05). (E) Correlation Megakaryocytes/Spleen changes: the line graph indicates the mean quantity of megakaryocytes/field, where continuous straight line represents the value found in G1 (healthy). The ordinate on the left indicates the numerical amount of megakaryocytes. The bar graph in green color indicates the variations of commitments of spleen changes found in the groups studied. The ordinate on the right indicates the score of histological changes observed in the spleen cuts, where the number 0 indicates no change, and the number 5 indicates maximum change. The groups are represented as follows: G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30x pre-treated), G4 (BioLi30 pre/post-treated), G5 (BioLi30 post-treated), G6 (Water 30 post-treated), G7 (Antimonium crudum 30 post-treated) and G8 (GlucantimeÒ post-treated).
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The percentage of megakaryocytes and their relationship with spleen changes are shown in Fig. 1D and E, respectively. 3.2. Cytokines and Th1/Th2 ratio A decrease in IFN-c and IL-12 concentrations in G2 (L. infantum) was observed when compared to G1 (healthy). Moreover, an increase occurred with IL4 and IL-10 concentrations, as expected in the L. infantum infection. Comparing G1 with treated groups, there was no significant difference in IFN-c cytokine (p > 0.05), but there was for IL-4 in G8, IL-10 in G6, and IL-12 in G5 (p < 0.05). Regarding the comparison among G2 and treated groups, there was a significant difference (p < 0.05) with G5 in IFN-c, G8 in IL-4, as well as G5 and G6 in IL-12. IL-10 production had no significant difference between G2 and treated groups (p > 0.05). However, it was observed a decreasing of this cytokine in G4. The IFN-c/IL4 ratio analysis revealed a significant difference in G1 with G6 and G8 (p < 0.05), as well as between G2 and G6 (p < 0.05). No statistical differences were observed in the IFN-c/ IL-10 ratio (p > 0.05) between all analyzed groups (Fig. 2). 4. Discussion It is known that megakaryocytes are related to L. infantum infection resistance and they increase in an attempt to control the disease [5,9,11]. The parasite can induce replication of these cells in the spleen and, due to their immaturity, the infection will be enhanced [9,11]. This information corroborates Fig. 1D and E data, due to the higher number of cells observed in all infected groups, except in G7, where megakaryocytes diminishing could be correlated to anti-inflammatory response, and to the inhibition of mononuclear cell migration to the injury site, as previously described [5]. These effects in animals with leishmaniasis probably
occur in parallel to the inhibition of megakaryocytes expansion in spleen. Fig. 2 showed Th1 predominance in G1 (uninfected control) when compared to G2 (infected but untreated control), by IFN-c increase concomitant with IL-4 and IL-10 decreasing. BALB/c mice are susceptible to L. infantum [15]. Therefore, Th2 response observed in G2 confirms this fact, since it was related to a worst prognosis [5]. Maybe G2 megakaryocytes increase could be an immune system attempt to create L. infantum infection resistance [9,11]. Regarding the analyzed cytokines in this study (Fig. 2), the increase of IFN-c concentration in G5 (BioLi30 post-treated) may be related to the polyclonal activation of B-cells, leading to antibody production and raising levels of IgM and IgG [8]. However, it was observed, within this dynamic, that both G4 (BioLi30 pre/post-treated) and G7 (Antimonium crudum 30 post-treated) were able to increase this cytokine, in order to return to G1 (healthy animals) basal levels. By positive feedback mechanism, the results on IL-12 were similar to IFN-c. In IL-4, the raising of this cytokine concentration in G8 (GlucantimeÒ post-treated) may be related to a cytotoxic effect caused by GlucantimeÒ [1], since the tissue damage will activate T lymphocytes and will increase the IgE production [10]. In IL-10, the raising levels observed in G6 (Water 30 post-treated) may be related to the negative feedback mechanism occurred between this cytokine and IL-12/IFN-c. Additionally, IFN-c has also increased in this same group. These findings indicated water effects were unspecific, probably due to the process of homeopathic dynamization that may form aggregates known as ‘‘nanobubbles” or ‘‘clusters”, which make immunomodulatory reactions. The IL-10 diminishing in G4 may probably indicate that smaller macrophage activation occurred with the pre/ post-treatment (promoted by BioLi30X), which induced a better immune defense to the animals of this group. Whereas, we cannot say which group caused greater or less resistance to the host,
Fig. 2. Dosage of cytokines and Th1/Th2 ratio. The groups are represented as follows: G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30 pre-treated), G4 (BioLi30 pre/post-treated), G5 (BioLi30 post-treated), G6 (Water 30 post-treated), G7 (Antimonium crudum 30 post-treated) and G8 (GlucantimeÒ post-treated). The solid lines indicate a statistically significant difference with the healthy animals group (G1), while the dashed ones indicate difference with untreated L. infantum infected group (G2). Among treated groups (G3–G8) these differences were represented by ‘‘*” with the same corresponding letter, such as: (A) IFN-c: G5 presented IFN-c increased production when compared to G3, G4 and G7 (p < 0.01), and G8 (p < 0.05). (B) IL-4: G8 presented IL-4 increased production when compared to G3 (p < 0.05), G4, G5, G6 and G7 (p < 0.001). It was also observed difference between G3 and G6 (p < 0.01). (C) IL-10: G4 presented IL-10 decreased production, when compared to G3 (p < 0.05), G6 (p < 0.001) and G7 (p < 0.01). (D) IL-12: G5 presented IL-12 increased production when compared to G3, G4 and G7 (p < 0.001), and G8 (p < 0.01). It was also observed a significant difference between G6 with G3, and with G7 (p < 0.05). (E) Ratio IFN-c/IL-4: it was observed Th1 predominance in G4, G5, G6 and G7, and Th2 in G3 and G8. The group G6 showed increase when compared with G2, G3, G4, G5, G7 and G8 (p < 0.001); and G5 showed increase when compared with G3 and G8 (p < 0.05). (F) Correlation IFN-c/IL-10: it was observed Th1 predominance in G4, G5, G7 and G8, and Th2 in groups G3 and G6, with no significant difference among all analyzed groups (p > 0.05). Statistical differences were revealed by parametric One way ANOVA – Tukey test.
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exclusively by the individual dosages of cytokines, making necessary the analysis of Th1/Th2 ratio, as below described. In G3 (BioLi30 pre-treated), probably the oral immunization promoted by prior treatment could have led to a humoral immune response against the parasite antigens, enhancing Th2 cytokines. In G6 (Water 30 post-treated), although Th1 cytokine predominance has occurred, only by increased IFN-c in the ratio IFN-c/IL4, the treatment in question was not able to control megakaryocytes elevation and presented the highest level of spleen changes (Fig. 1), staying just below G2 (infected with L. infantum) and G8 (GlucantimeÒ post-treated). In G8, this Th2 predominance, as well as spleen changes and elevation of megakaryocytes, probably occurred due to the cytotoxicity of GlucantimeÒ. Regarding G4 (BioLi30 pre/post-treated), G5 (BioLi30 post-treated) and G7 (Antimonium crudum 30 post-treated), changes in Th1/Th2 ratio were observed, verifying predominance of Th1 cytokine. However, comparing G4 with G5, the second one caused higher megakaryocytes production and spleen changes. The comparison between G4 and G7 showed that both groups had equal levels of white pulp hyperplasia with spleen disruption, and expressed the lowest megakaryocytes concentration, indicating systemic inflammation reduction. In conclusion, BioLi30 pre/post-treatment and Antimonium crudum 30 post-treatment were better able to contain the disease, suggesting they can modulate the immune system toward as better prognosis (Th1 response), as described in other works with homeopathic medicines [4,5,7]. However, the clinical significance of these findings needs to be further investigated. Funding information This study was financed by CAPES. Conflict of interests The authors have no conflict of interest to declare. Ethical approval The study was approved by the Standing Committee on Ethics in Research Involving Animals, according to CEUA 66/2014. Author’s contributions Ana Paula Bacellar Cajueiro: designed the research plan, organized the study, participated in all experiments, coordinated the data-analysis and drafted this manuscript. Ester Puna Goma: helped in animals’ infection, in their treatments and in euthanasia. Hilton Antônio Mata dos Santos: helped in animals’ blood collection and in euthanasia. Igor Almeida Rodrigues: provided the L. infantum strain, helped in animals’ infection and interpreted the data of this manuscript. Helena Keiko Toma: interpreted the data of this manuscript.
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Silvana Marques Araújo: helped to draft, interpreted the cytokines data and corrected this manuscript. Leoni Villano Bonamin: analyzed the histological cuts and megakaryocytes changes, interpreted the data and revised this manuscript. Nelson Brêtas de Noronha Gomes: analyzed the histological cuts and megakaryocytes changes. Morgana Teixeira Castelo-Branco: helped to develop the cytokine analysis and interpret and correct their data. Edilma Paraguai de Souza Dias: helped to do animals’ histological cuts and in euthanasia. Alexandre dos Santos Pyrrho: designed the research plan, organized the study, coordinated the data-analysis, and interpreted and corrected this manuscript. Carla Holandino Quaresma: designed the research plan, organized the study, coordinated the data-analysis, and interpreted and corrected this manuscript. References [1] Brasil, Manual de Vigilância e Controle da Leishmaniose Visceral, Ministério da Saúde, Brasília: Editora MS, ed. 1, 2006. [2] F.N. Ferraz, G.K. Simoni, S.M. de Araújo, et al., Different forms of administration of biotherapy 7DH in mice experimentally infected by Trypanosoma cruzi produce different effects, Homeopathy 100 (2011) 237–243. [3] P.F. Sandri, G.J.S. Falkowski, S.M. de Araújo, et al., Biotherapic of Trypanosoma cruzi 17 controlled histopathological alterations in mice infected by this protozoon, Int. J. High Dilut. Res. 10 (36) (2011) 110–114. [4] G.J. Falkowski-Temporini, C.R. Lopes, P.F. Massani, et al., Predominance of Th1 response, increase of megakaryocytes and Kupffer cells are related to survival in Trypanosoma cruzi infected mice treated with Lycopodium clavatum, Cytokine 88 (2016) 57–61. [5] F.R. de Santana, C.de P. Coelho, L.V. Bonamin, et al., Modulation of inflammation response to murine cutaneous Leishmaniasis by homeopathic medicines: Antimonium crudum 30cH, Homeopathy (103) (2014) 264–274. [6] Brasil, Farmacopeia Homeopática Brasileira, Ministério da Saúde, ed. 3, 2011. [7] C.M. Siqueira, P.D. Motta, C. Holandino, et al., Homeopathic treatments modify inflammation but not behavioral response to influenza antigen challenge in BALB/c mice, Homeopathy 105 (2016) 257–264. [8] A. Nieto, G. Domínguez-Bernal, J. Carríon, et al., Mechanisms of resistance and susceptibility to experimental visceral leishmaniosis: BALB/c mouse versus Syrian hamster model, Veter. Res. 42 (39) (2011) 1–13. [9] J. Carríon, S. Iborra, C. Alonso, et al., Immunohistological features of visceral leishmaniasis in BALB/c mice, Paras. Immunol. 28 (2006) 173–183. [10] A.K. Abbas e A.H. Lichtman, Imunologia Básica - funções e distúrbios do sistema imunológico, vol. 4, Elsevier, Rio de Janeiro, 2013. [11] C.G. Pereira, A.L.N. Silva, M.A. Souza, et al., Different isolates from Leishmania braziliensis complex induce distinct histopathological features in a murine model of infection, Veter. Parasitol. 165 (2009) 231–240. [12] R.C.Z. Nascimento, Determinação da sensibilidade de isolados de Leishmania a Antimoniato de meglumina, Anfotericina B e Tamoxifeno, Dissertation presented to the parasitology department at the University of São Paulo, Biology Program Relationship Host-Pathogen, São Paulo, 2009. [13] F.H. Rodrigues, S.R. Afonso-Cardoso, M.A. Souza, et al., Effect of imidocarb and levamisole on the experimental infection of BALB/c mice by Leishmania (Leishmania) amazonensis, Veter. Parasitol.gy 139 (2006) 37–46. [14] D.C. Miguel, R.C. Zauli-Nascimento, J.K.U. Yokoyama-Yasunaka, et al., Tamoxifen as a potencial antileishmanial agent: efficacy in the treatment of Leishmania braziliensis and Leishmania chagasi infections, J. Antimicrob. Chemother. 63 (2) (2008) 365–368. [15] S. Ahmed, M. Colmenares, L. Soong, et al., Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis, Infect. Immun. 71 (1) (2003) 410.
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Short communication
Homeopathic medicines cause Th1 predominance and induce spleen and megakaryocytes changes in BALB/c mice infected with Leishmania infantum Ana Paula Bacellar Cajueiro a,1, Ester Puna Goma a,2, Hilton Antônio Mata dos Santos a,2, Igor Almeida Rodrigues a,3, Helena Keiko Toma a,4, Silvana Marques Araújo b,5, Leoni Villano Bonamin c,6, Nelson Brêtas de Noronha Gomes d,7, Morgana Teixeira Lima Castelo-Branco e,8, Edilma Paraguai de Souza Dias f,9, Alexandre dos Santos Pyrrho a,2, Carla Holandino a,⇑ a
Graduation Program in Pharmaceutical Sciences, Universidade Federal do Rio de Janeiro, Brazil Department of Health Sciences, Universidade Estadual de Maringá, Brazil Graduation Program of Environmental and Experimental Pathology, Universidade Paulista, Brazil d Department of Veterinary Medicine, Universidade Federal de Lavras, Brazil e Biomedical Sciences Institute, Universidade Federal do Rio de Janeiro, Brazil f Graduation Program in Biological Sciences, Universidade Federal do Rio de Janeiro, Brazil b c
a r t i c l e
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Article history: Received 31 October 2016 Received in revised form 9 February 2017 Accepted 12 February 2017
Keywords: Leishmania infantum Biotherapy Antimonium crudum
a b s t r a c t The prevalence of Th1/Th2 response, spleen changes and megakaryocytes were investigated in BALB/c mice (n = 138) infected with Leishmania infantum, and treated with Leishmania infantum 30 (10 30) biotherapy – BioLi30. We performed controlled experiments using 8-to-12-week-old mice, infected with 5 107 L. infantum promastigotes, divided into eight groups: G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30 pre-treated), G4 (BioLi30 pre/post-treated), G5 (BioLi30 post-treated), G6 (Water 30 post-treated), G7 (Antimonium crudum 30 post-treated) and G8 (GlucantimeÒ post-treated). G3 –G7 groups were orally treated with their respective drugs diluted in filtered water (1:10), and G8 received GlucantimeÒ (0.6 mg/100 ml of PBS), intraperitoneally. Spleen fragments were submitted to double blind histopathological evaluation and the number of megakaryocytes was counted. Besides, animals’ serum
Abbreviations: Th1, T helper cell type 1; Th2, T helper cell type 2; IFN-c, Interferon gama; IL-4, Interleukin 4; IL-10, Interleukin 10; IL-12, Interleukin 12; AVL, American visceral leishmaniasis; BioLi30, Leishmania infantum 30 (10 30). ⇑ Corresponding author at: Laboratório Multidisciplinar de Ciências Farmacêuticas e Laboratório de Pesquisa e Desenvolvimento em Práticas Integrativas e Complementares, Departamento de Fármacos e Medicamentos, Faculdade de Farmácia, Bloco B, ss 11 e 34., Av. Carlos Chagas Filho 373, CEP: 21941-902, Centro de Ciências da Saúde (CCS), Cidade Universitária, Ilha do Fundão, UFRJ – Rio de Janeiro, RJ, Brazil. E-mail addresses: [email protected] (A.P.B. Cajueiro), [email protected] (E.P. Goma), [email protected] (H.A.M. dos Santos), [email protected] (I. Almeida Rodrigues), [email protected] (H.K. Toma), [email protected] (S.M. Araújo), [email protected] (L.V. Bonamin), [email protected] (N.B.N. Gomes), [email protected] (M.T.L. Castelo-Branco), [email protected] (E.P. de Souza Dias), [email protected] (A. dos Santos Pyrrho), [email protected] (C. Holandino). 1 Address: Graduation Program in Pharmaceutical Sciences, Multidisciplinary Laboratory of Pharmaceutical Sciences, Department of Drugs and Medicines, Faculty of Pharmacy, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco B-ss, salas 11 e 34, 21941-590 Rio de Janeiro, RJ, Brazil. 2 Address: Graduation Program in Pharmaceutical Sciences, Laboratory of Immuno-parasitology and Toxicological Analysis, Department of Social Hygiene and Clinical Analysis, Faculty of Pharmacy, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco A, sala 17, 21941-590, Brazil. 3 Address: Graduation Program in Pharmaceutical Sciences, Laboratory of Antimicrobials Prospection, Department of Natural Products and Food, Faculty of Pharmacy, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco A-ss, sala 14, 21941-590, Brazil. 4 Address: Graduation Program in Pharmaceutical Sciences, Laboratory of Molecular Diagnosis and Hematology, Department of Clinical and Toxicology Analysis, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco A, sala 13, 21941-590, Brazil. 5 Address: Department of Basic Health Sciences, Laboratory of Parasitology, Universidade Estadual de Maringá, Av Colombo 5790, Bloco I-90, sala 11, KM 130, 87020-900 Maringá, PR, Brazil. 6 Address: Graduation Program of Environmental and Experimental Pathology, Research Center of University Paulista, Department of General Pathology and Pathogenesis of Susceptibility, Universidade Paulista, Rua Dr. Bacelar 1212, 4° andar, 04026-002 São Paulo, SP, Brazil. 7 Address: Department of Veterinary Medicine, Animal Pathology Sector, Universidade Federal de Lavras, Campus Universitário, 37200-000 Minas Gerais, MG, Brazil. 8 Address: Laboratory of Cellular Immunology, Biomedical Science Institute, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco B, sala 19, 21941590, Brazil. 9 Address: Graduation Program in Biological Sciences, Bioinovar Laboratory: Unit of Bioproducts, Biocatalysis and Bioprocess, Paulo de Góes’ Microbiology Institute, Universidade Federal do Rio de Janeiro, UFRJ, Av. Carlos Chagas Filho 373, Bloco I, sala 31, 21941-390, Brazil. http://dx.doi.org/10.1016/j.cyto.2017.02.010 1043-4666/Ó 2017 Elsevier Ltd. All rights reserved.
98 Megakaryocytes BALB/c Th1/Th2 response
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was measured after 49 days of infection, and cytokines (IFN-c, IL-4, IL-10, IL-12), as well as the Th1/Th2 correlation (IFN-c/IL-4 and IFN-c/IL-10), were analyzed. Spleen histological parameters were classified as: healthy appearance (G1); discreet (G3–G7), moderate (G2) and moderate to severe (G8) white pulp hyperplasia; proliferation of megakaryocytes (G2–G8), and intense disruption (G2–G8). All groups, except for G7, showed higher percentages of megakaryocytes per field ranging from 87% to 15%, when compared to healthy animals (G1). Th1 predominance in IFN-c/IL-4 ratio (comparing to G2) was detected in G4, G5, G6 and G7. Finally, pre/post (BioLi30x) and post-treatment (Antimonium crudum 30x) presented reduction of megakaryocytes/spleen changes due to immunomodulation animal process, controlling the infection process, probably by the Th1 cytokine predominance. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction American visceral leishmaniasis (AVL) is a serious public health problem [1]. It is a worldwide neglected disease caused by Leishmania (L.) infantum, an intracellular parasite of the mononuclear phagocyte system cells, traditionally treated with GlucantimeÒ [1]. Several studies have demonstrated immunomodulatory effects of homeopathic medicines on diseases caused by Leishmania sp. and other parasites [2–5]. Considering these promising homeopathic results, we prepared Biotherapics, following the homeopathic pharmaceutical procedure [6], and verified their efficacy. As control, we used Antimonium crudum 30, since this medication covers almost leishmaniasis symptoms totality and has been described in other works [5,7]. Thus, the therapeutic and immunomodulatory biotherapy potential [2,3,7] motivated the present work in which the efficacy of a new biotherapy prepared with infective Leishmania infantum promastigotes (BioLi30) was evaluated in animal models. In the ALV disease, there is a predominance of Th2 CD4+ lymphocytes that will produce specific cytokines, increasing IL-4 and IL-10 due to the decrease of IL-12 and IFN-c (Th1-type cytokines), for a negative feedback mechanism [8]. Therefore, it is common to observe the predominance of Th2type cytokines in infected animals and, in contrast, Th1 in treated animals, conferring infection resistance in the last one [8]. These changes in Th1/Th2 relation can be clearly observed in BALB/c mice, as they are an excellent model to evaluate the disease’s immunomodulatory effects [8]. Spleens of BALB/c infected by L. infantum presented hypertrophy and hyperplasia of the mononuclear phagocyte system, accompanied by white pulp organ’s lymphoid depletion [9]. In this organ, it is common to observe megakaryocytes, a platelet precursor cells [10], whose numerical increase has been considerably shown in some works [9,11], indicating hematopoietic activity accompanied by a parasite expansion, taking to serious spleen functional disruptions [10,11]. Thus, when tissue injury occurs, the organism tries to increase the production of platelets, since they will be responsible for maintaining the capillaries integrity [10]. In addition, platelets were recently recognized in the immune response activation, as elements involved in the elimination of pathogenic agents [4]. Therefore, the aim of this study is to evaluate the prevalence of Th1/Th2 response, histological spleen changes and megakaryocytes participation in immunomodulation process of BALB/c mice with AVL, treated with BioLi30. 2. Material and methods
kept under controlled temperature and lighting conditions, with light/dark cycle (12 h/22 ± 1 °C). 2.2. Test solutions and experimental groups BioLi30x was prepared from a suspension containing 5x107 infective promastigotes of L. infantum/mL (strain MHOM/ BR/1974/PP75 - CLIOC/Fiocruz/RJ) at a ratio of 1:10, following the Brazilian Homeopathic Pharmacopoeia [6]. To prepare the dilutions, we used sterile phosphate buffer saline (PBS) from 1 (10 1) to 6 (10 6) potencies, since our preliminary experiments indicated parasite lyses when dynamization process was done with water as solvent up to 3. To ensure the protozoa integrity, PBS was used up to 6. After this, 7 (10 7) to 30 (10 30) potencies were prepared with sterile distilled water. For dynamizations, the diluted solutions were submitted to one hundred mechanical succussions in Homeopathic Brazilian Machine (DeniseÒ-AUTIC). This procedure was performed because several works showed the biological effects of hydro-alcoholic dynamized solvents [2–5,7]. Besides, considering previous homeopathic uses of Antimonium crudum [5], we also evaluated its effects (Antimonium crudum 30) in comparison to the traditional medicine GlucantimeÒ (allopathic control). The GlucantimeÒ (Sanofi-Aventis, lot 019106, 300 mg/mL) was prepared at 0.6 mg/100 mL of sterile phosphate buffer saline (PBS) [12]. BALB/c mice were randomly divided into eight groups (n = 16–18): G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30 pre-treated), G4 (BioLi30 pre/post-treated), G5 (BioLi30 posttreated), G6 (Water 30 post-treated), G7 (Antimonium crudum 30 post-treated) and G8 (GlucantimeÒ post-treated). 2.3. Infection and treatment schemes Except for G1, all groups were infected with L. infantum promastigotes. Parasites were previously maintained in PBHIL medium supplemented with 10% of fetal bovine serum (FBS) at 27 °C [13]. At the end of log phase (120 h of growth), the cells were harvest, centrifuged (3500 rpm/5 min) and washed twice with PBS at pH 7.2. The mice were challenged with a suspension of 5 107 promastigotes/100 mL in sterile PBS, intraperitoneally. The animals were treated orally (G3–G7 groups) and intraperitoneally (G8). G3 and G4 were pre-treated for 21 days. At the end of this period, all groups were infected and, to ensure the disease establishment, there was a pause of 28 days prior the posttreatment beginning of G4–G8 for more 21 days [14].
2.1. Experimental design 2.4. Euthanasia and collection of biological materials Double blind controlled experiments using BALB/c mice (n = 138), with 8-to-12-week-old were performed. All animals were acquired at the Reference Center and Mice creation for Scientific Research of UFRJ after ethics committee approval (CEUA/UFRJ066/14). Water and feed were provided ad libitum and the animals
After 49 days of infection, mice were anesthetized with chlorhydrates of ketamine (100–150 mg/kg) and xylazine (10–15 mg/kg), intramuscularly. Sequentially, 800 lL of blood were collected by cardiac puncture and the serum centrifuged at
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10.000 rpm/10 min in centrifugal Eppendorf 5415-C, for cytokine analyzes. Euthanasia was performed by exposure to increasing CO2 atmosphere. Spleen fragments were removed, fixed in blade with 4% paraformaldehyde, and stained with hematoxylin-eosin to perform histopathological changes. The slides were observed by two independent veterinarian pathologists to avoid interpretation bias. All experimental procedures involving animals were approved by Ethics Committee (CEUA) of UFRJ, under the protocol number 066/14. 2.5. Spleen changes and quantification of megakaryocytes Histological spleen cuts were observed in optical microscope (Axyoplan 2, Zeiss/German). The spleen changes were sorted by score (0–5), indicating (in ascending order) the degree of white pulp hyperplasia with spleen disruption. The megakaryocytes were counted in six random fields. The average found in the groups were compared with the uninfected control (G1) and expressed in percentage.
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phage functions dependent of IFN-c, and IL-10 inhibits the production of IL-12) were measured in serum (ng/mL) in three/four pools at the 49th day of infection. Samples were analyzed in duplicate in ELISA 96-well plates (Costar 3590 Corning), using the commercial kit R&D Systems (USA), and read in Elisa Bio-Rad plate reader at 450 nm. Sequentially, the ratio averages of Th1/Th2 (IFN-c/IL-4 and IFN-c/IL-10) were determined by dividing the values obtained in Th1 cytokine by Th2 cytokines.
2.7. Statistical analysis One-way ANOVA was performed, followed by Tukey test to evaluate parametric variables (cytokines) and Kruskal-Wallis followed by Dunn test were performed to evaluate non-parametric variables (megakaryocytes). Graphpad Prism 5Ò software was used. P values < 0.05 were considered statistically significant.
3. Results 2.6. Cytokines and ratio Th1/Th2 The cytokines IFN-c (pro-inflammatory which activates macrophages in order to inhibit infection and modulates B lymphocytes activities: Th1 response), IL-12 (induces T CD4+ cell differentiation, which is responsible for Th1 cytokines expansion), IL4 and IL-10 (anti-inflammatories with Th2 response: IL-4 suppresses macro-
3.1. Spleen changes and quantification of megakaryocytes Spleen histological cuts presented: healthy appearance (G1); discreet (G3–G7), moderate (G2) and moderate to severe (G8; Fig. 1A) white pulp hyperplasia; proliferation of and megakaryocytes (G2–G8); and intense disruption (G2–G8; Fig. 1B and C).
Fig. 1. Spleen changes and quantification of megakaryocytes. (A) Spleen histological cuts, indicating moderate to severe white pulp hyperplasia (G2 and G8). (B) Spleen histological cuts indicating severe organ disruption (G2 and G8). (C) in set, indicating presence of severe quantity of megakaryocytes. (D) Percentage of megakaryocytes: calculated dividing the groups’ averages by G1 average (healthy animals). Statistical differences were revealed by non-parametric Kruskal-Wallis/Dunn, indicating that only G7 animals showed 10% of percentage reduction (p > 0.05) when compared to G1. All other groups showed higher values than those recorded in G1: G2 (87%; p < 0.001), G8 (53%; p < 0.001), G6 (39%; p < 0.001), G3 (29%; p < 0.05), G5 (23%; p > 0.05) and G4 (15%; p > 0.05). Compared to G2 (L. infantum), a statistically significant difference was found between this group and G3, G4, G5 and G7 (p < 0.001). Among treated groups, the following p-values were found: G7 with G3, G4, G5, G6 and G8 (p < 0.001); G4 with G6 and G8 (p < 0.01), and G5 with G6 and G8 (p < 0.05). (E) Correlation Megakaryocytes/Spleen changes: the line graph indicates the mean quantity of megakaryocytes/field, where continuous straight line represents the value found in G1 (healthy). The ordinate on the left indicates the numerical amount of megakaryocytes. The bar graph in green color indicates the variations of commitments of spleen changes found in the groups studied. The ordinate on the right indicates the score of histological changes observed in the spleen cuts, where the number 0 indicates no change, and the number 5 indicates maximum change. The groups are represented as follows: G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30x pre-treated), G4 (BioLi30 pre/post-treated), G5 (BioLi30 post-treated), G6 (Water 30 post-treated), G7 (Antimonium crudum 30 post-treated) and G8 (GlucantimeÒ post-treated).
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The percentage of megakaryocytes and their relationship with spleen changes are shown in Fig. 1D and E, respectively. 3.2. Cytokines and Th1/Th2 ratio A decrease in IFN-c and IL-12 concentrations in G2 (L. infantum) was observed when compared to G1 (healthy). Moreover, an increase occurred with IL4 and IL-10 concentrations, as expected in the L. infantum infection. Comparing G1 with treated groups, there was no significant difference in IFN-c cytokine (p > 0.05), but there was for IL-4 in G8, IL-10 in G6, and IL-12 in G5 (p < 0.05). Regarding the comparison among G2 and treated groups, there was a significant difference (p < 0.05) with G5 in IFN-c, G8 in IL-4, as well as G5 and G6 in IL-12. IL-10 production had no significant difference between G2 and treated groups (p > 0.05). However, it was observed a decreasing of this cytokine in G4. The IFN-c/IL4 ratio analysis revealed a significant difference in G1 with G6 and G8 (p < 0.05), as well as between G2 and G6 (p < 0.05). No statistical differences were observed in the IFN-c/ IL-10 ratio (p > 0.05) between all analyzed groups (Fig. 2). 4. Discussion It is known that megakaryocytes are related to L. infantum infection resistance and they increase in an attempt to control the disease [5,9,11]. The parasite can induce replication of these cells in the spleen and, due to their immaturity, the infection will be enhanced [9,11]. This information corroborates Fig. 1D and E data, due to the higher number of cells observed in all infected groups, except in G7, where megakaryocytes diminishing could be correlated to anti-inflammatory response, and to the inhibition of mononuclear cell migration to the injury site, as previously described [5]. These effects in animals with leishmaniasis probably
occur in parallel to the inhibition of megakaryocytes expansion in spleen. Fig. 2 showed Th1 predominance in G1 (uninfected control) when compared to G2 (infected but untreated control), by IFN-c increase concomitant with IL-4 and IL-10 decreasing. BALB/c mice are susceptible to L. infantum [15]. Therefore, Th2 response observed in G2 confirms this fact, since it was related to a worst prognosis [5]. Maybe G2 megakaryocytes increase could be an immune system attempt to create L. infantum infection resistance [9,11]. Regarding the analyzed cytokines in this study (Fig. 2), the increase of IFN-c concentration in G5 (BioLi30 post-treated) may be related to the polyclonal activation of B-cells, leading to antibody production and raising levels of IgM and IgG [8]. However, it was observed, within this dynamic, that both G4 (BioLi30 pre/post-treated) and G7 (Antimonium crudum 30 post-treated) were able to increase this cytokine, in order to return to G1 (healthy animals) basal levels. By positive feedback mechanism, the results on IL-12 were similar to IFN-c. In IL-4, the raising of this cytokine concentration in G8 (GlucantimeÒ post-treated) may be related to a cytotoxic effect caused by GlucantimeÒ [1], since the tissue damage will activate T lymphocytes and will increase the IgE production [10]. In IL-10, the raising levels observed in G6 (Water 30 post-treated) may be related to the negative feedback mechanism occurred between this cytokine and IL-12/IFN-c. Additionally, IFN-c has also increased in this same group. These findings indicated water effects were unspecific, probably due to the process of homeopathic dynamization that may form aggregates known as ‘‘nanobubbles” or ‘‘clusters”, which make immunomodulatory reactions. The IL-10 diminishing in G4 may probably indicate that smaller macrophage activation occurred with the pre/ post-treatment (promoted by BioLi30X), which induced a better immune defense to the animals of this group. Whereas, we cannot say which group caused greater or less resistance to the host,
Fig. 2. Dosage of cytokines and Th1/Th2 ratio. The groups are represented as follows: G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30 pre-treated), G4 (BioLi30 pre/post-treated), G5 (BioLi30 post-treated), G6 (Water 30 post-treated), G7 (Antimonium crudum 30 post-treated) and G8 (GlucantimeÒ post-treated). The solid lines indicate a statistically significant difference with the healthy animals group (G1), while the dashed ones indicate difference with untreated L. infantum infected group (G2). Among treated groups (G3–G8) these differences were represented by ‘‘*” with the same corresponding letter, such as: (A) IFN-c: G5 presented IFN-c increased production when compared to G3, G4 and G7 (p < 0.01), and G8 (p < 0.05). (B) IL-4: G8 presented IL-4 increased production when compared to G3 (p < 0.05), G4, G5, G6 and G7 (p < 0.001). It was also observed difference between G3 and G6 (p < 0.01). (C) IL-10: G4 presented IL-10 decreased production, when compared to G3 (p < 0.05), G6 (p < 0.001) and G7 (p < 0.01). (D) IL-12: G5 presented IL-12 increased production when compared to G3, G4 and G7 (p < 0.001), and G8 (p < 0.01). It was also observed a significant difference between G6 with G3, and with G7 (p < 0.05). (E) Ratio IFN-c/IL-4: it was observed Th1 predominance in G4, G5, G6 and G7, and Th2 in G3 and G8. The group G6 showed increase when compared with G2, G3, G4, G5, G7 and G8 (p < 0.001); and G5 showed increase when compared with G3 and G8 (p < 0.05). (F) Correlation IFN-c/IL-10: it was observed Th1 predominance in G4, G5, G7 and G8, and Th2 in groups G3 and G6, with no significant difference among all analyzed groups (p > 0.05). Statistical differences were revealed by parametric One way ANOVA – Tukey test.
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exclusively by the individual dosages of cytokines, making necessary the analysis of Th1/Th2 ratio, as below described. In G3 (BioLi30 pre-treated), probably the oral immunization promoted by prior treatment could have led to a humoral immune response against the parasite antigens, enhancing Th2 cytokines. In G6 (Water 30 post-treated), although Th1 cytokine predominance has occurred, only by increased IFN-c in the ratio IFN-c/IL4, the treatment in question was not able to control megakaryocytes elevation and presented the highest level of spleen changes (Fig. 1), staying just below G2 (infected with L. infantum) and G8 (GlucantimeÒ post-treated). In G8, this Th2 predominance, as well as spleen changes and elevation of megakaryocytes, probably occurred due to the cytotoxicity of GlucantimeÒ. Regarding G4 (BioLi30 pre/post-treated), G5 (BioLi30 post-treated) and G7 (Antimonium crudum 30 post-treated), changes in Th1/Th2 ratio were observed, verifying predominance of Th1 cytokine. However, comparing G4 with G5, the second one caused higher megakaryocytes production and spleen changes. The comparison between G4 and G7 showed that both groups had equal levels of white pulp hyperplasia with spleen disruption, and expressed the lowest megakaryocytes concentration, indicating systemic inflammation reduction. In conclusion, BioLi30 pre/post-treatment and Antimonium crudum 30 post-treatment were better able to contain the disease, suggesting they can modulate the immune system toward as better prognosis (Th1 response), as described in other works with homeopathic medicines [4,5,7]. However, the clinical significance of these findings needs to be further investigated. Funding information This study was financed by CAPES. Conflict of interests The authors have no conflict of interest to declare. Ethical approval The study was approved by the Standing Committee on Ethics in Research Involving Animals, according to CEUA 66/2014. Author’s contributions Ana Paula Bacellar Cajueiro: designed the research plan, organized the study, participated in all experiments, coordinated the data-analysis and drafted this manuscript. Ester Puna Goma: helped in animals’ infection, in their treatments and in euthanasia. Hilton Antônio Mata dos Santos: helped in animals’ blood collection and in euthanasia. Igor Almeida Rodrigues: provided the L. infantum strain, helped in animals’ infection and interpreted the data of this manuscript. Helena Keiko Toma: interpreted the data of this manuscript.
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Silvana Marques Araújo: helped to draft, interpreted the cytokines data and corrected this manuscript. Leoni Villano Bonamin: analyzed the histological cuts and megakaryocytes changes, interpreted the data and revised this manuscript. Nelson Brêtas de Noronha Gomes: analyzed the histological cuts and megakaryocytes changes. Morgana Teixeira Castelo-Branco: helped to develop the cytokine analysis and interpret and correct their data. Edilma Paraguai de Souza Dias: helped to do animals’ histological cuts and in euthanasia. Alexandre dos Santos Pyrrho: designed the research plan, organized the study, coordinated the data-analysis, and interpreted and corrected this manuscript. Carla Holandino Quaresma: designed the research plan, organized the study, coordinated the data-analysis, and interpreted and corrected this manuscript. References [1] Brasil, Manual de Vigilância e Controle da Leishmaniose Visceral, Ministério da Saúde, Brasília: Editora MS, ed. 1, 2006. [2] F.N. Ferraz, G.K. Simoni, S.M. de Araújo, et al., Different forms of administration of biotherapy 7DH in mice experimentally infected by Trypanosoma cruzi produce different effects, Homeopathy 100 (2011) 237–243. [3] P.F. Sandri, G.J.S. Falkowski, S.M. de Araújo, et al., Biotherapic of Trypanosoma cruzi 17 controlled histopathological alterations in mice infected by this protozoon, Int. J. High Dilut. Res. 10 (36) (2011) 110–114. [4] G.J. Falkowski-Temporini, C.R. Lopes, P.F. Massani, et al., Predominance of Th1 response, increase of megakaryocytes and Kupffer cells are related to survival in Trypanosoma cruzi infected mice treated with Lycopodium clavatum, Cytokine 88 (2016) 57–61. [5] F.R. de Santana, C.de P. Coelho, L.V. Bonamin, et al., Modulation of inflammation response to murine cutaneous Leishmaniasis by homeopathic medicines: Antimonium crudum 30cH, Homeopathy (103) (2014) 264–274. [6] Brasil, Farmacopeia Homeopática Brasileira, Ministério da Saúde, ed. 3, 2011. [7] C.M. Siqueira, P.D. Motta, C. Holandino, et al., Homeopathic treatments modify inflammation but not behavioral response to influenza antigen challenge in BALB/c mice, Homeopathy 105 (2016) 257–264. [8] A. Nieto, G. Domínguez-Bernal, J. Carríon, et al., Mechanisms of resistance and susceptibility to experimental visceral leishmaniosis: BALB/c mouse versus Syrian hamster model, Veter. Res. 42 (39) (2011) 1–13. [9] J. Carríon, S. Iborra, C. Alonso, et al., Immunohistological features of visceral leishmaniasis in BALB/c mice, Paras. Immunol. 28 (2006) 173–183. [10] A.K. Abbas e A.H. Lichtman, Imunologia Básica - funções e distúrbios do sistema imunológico, vol. 4, Elsevier, Rio de Janeiro, 2013. [11] C.G. Pereira, A.L.N. Silva, M.A. Souza, et al., Different isolates from Leishmania braziliensis complex induce distinct histopathological features in a murine model of infection, Veter. Parasitol. 165 (2009) 231–240. [12] R.C.Z. Nascimento, Determinação da sensibilidade de isolados de Leishmania a Antimoniato de meglumina, Anfotericina B e Tamoxifeno, Dissertation presented to the parasitology department at the University of São Paulo, Biology Program Relationship Host-Pathogen, São Paulo, 2009. [13] F.H. Rodrigues, S.R. Afonso-Cardoso, M.A. Souza, et al., Effect of imidocarb and levamisole on the experimental infection of BALB/c mice by Leishmania (Leishmania) amazonensis, Veter. Parasitol.gy 139 (2006) 37–46. [14] D.C. Miguel, R.C. Zauli-Nascimento, J.K.U. Yokoyama-Yasunaka, et al., Tamoxifen as a potencial antileishmanial agent: efficacy in the treatment of Leishmania braziliensis and Leishmania chagasi infections, J. Antimicrob. Chemother. 63 (2) (2008) 365–368. [15] S. Ahmed, M. Colmenares, L. Soong, et al., Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis, Infect. Immun. 71 (1) (2003) 410.