EFFECT OF AN IMMUNOMODULATING DIET ON THE IMMUNE SYSTEM OF DOGS INFECTED WITH Leishmania infantum

Transcription

1 EFFECT OF AN IMMUNOMODULATING DIET ON THE IMMUNE SYSTEM OF DOGS INFECTED WITH Leishmania infantum Summary The experiment conducted on the effects of the nutraceutical food Immuno Active on dogs suffering from leishmaniasis suggests that this diet can, in the course of conventional therapy, modulate the immune system, stimulating the activity of cytotoxic T lymphocytes and the secretion of cytokines, and increasing regulatory T cells. These effects promote immune response against the parasite, reduce the infection load and reduce the inflammatory and immunopathogenic effects of the disease on the skin and internal organs. Immune response and its regulation The immune system has specific responses that include cellular responses, like the exertion of cytotoxic functions against infected cells, and humoral responses, such as complement, antibodies and cytokines. The cellular and humoral responses are two functional channels promoted by cytokine secretion by T helper cells. The secretory action of such cells can polarize the immune response towards pro-inflammatory action (Th1), which promotes cytotoxic functions, or anti-inflammatory action (Th2), which induces antibody production. Fine regulation of the immune response, as well as mechanisms of self tolerance, occur through a complex system of cellular and secretory interactions that include regulatory T cells (Treg) with phenotype CD3+CD4+FoxP3+ as the main player in the regulation. Tregs induce regulation through direct cell-cell interactions as well as the secretion of cytokines like IL-4, TGF-beta and IL-10. It is important to emphasize that the success of the immune system action in controlling infections is related to the establishment of a Th1 profile rather than a Th2 profile. In this respect, an efficacious immune response in the case of canine leishmaniasis (CL) is characterized by a proinflammatory cytokine profile (e.g. IFNgamma), while a Th2 profile (e.g. based on IL- 4 production) causes increased susceptibility to the disease and decreased response to 1

2 treatment in infected dogs. Little is known regarding the involvement of Treg cells in anti-leishmania immune response (de Lima et al., 2010; Cortese et al., 2011). The study of Immuno Active food as a dietary immunomodulator The objective of the present experimental research was to characterize the lymphocyte phenotype, cytokine profile and regulatory action of the immune system of dogs naturally infected with leishmaniasis undergoing conventional therapy (Nmethylglucamine antimoniate and allopurinol) in the absence or presence of dietary co-treatment with the nutriceutical food Immuno Active. The goal, therefore, was to determine the impact of Immuno Active, a diet with potential immunomodulatory activity, on the immune system of dogs naturally infected with L. infantum. It has been shown in numerous studies that the metabolic/nutritional condition of the subject can significantly modify the immune response and immune tolerance, as has been observed in both humans and animal models. In addition, it is known that malnutrition is one of the main risk factors for the onset of visceral leishmaniasis in dogs (Anstead et al., 2001; Malafaia et al., 2009). Duration of the study The study lasted a total of 12 months for each individual animal and was conducted between APRIL 2012 and JULY Subjects recruited The infected dogs were divided into two groups: Group I consisted of approximately 20 subjects treated with classic anti-leishmania therapy (meglumine antimoniate at a standard dosage of 100 mg/kg bw/day, divided into two daily doses of 50 mg/kg bw, by subcutaneous injection and allopurinol at a dosage of 10 mg/kg bw orally two times a day, all for 30 consecutive days). The allopurinol treatment for this group was not extended to 6-8 months, as is normally done, and the group was fed Immuno Active for 11 consecutive months instead of standard food. Group II, also composed of 20 dogs naturally infected with L. infantum, was treated with classic anti-leishmania therapy (meglumine antimoniate at a standard dosage of 100 mg/kg bw/day, divided into two daily doses of 50 mg/kg bw, by subcutaneous injection for 30 consecutive 2

3 days and allopurinol at a dosage of 10 mg/kg bw orally two times a day for 6-8 months). This group was fed the commercial food that the dog normally ate for 11 months. During a later stage of the experiment, Group III was created. These dogs, also naturally infected by L. infantum, were fed the Immuno Active diet but were not given any medicines. A Control Group was also created. It was composed of 30 healthy dogs (all middleaged, 5-6 years, 12 male and 18 female, to make the control group as homogenous as possible), with no clinical signs of leishmaniasis and negative results for serum, parasite and molecular tests. Clinical parameters and diagnostic procedures For each dog recruited, a detailed medical history was taken (no subject had undergone specific treatment for CL) and a thorough clinical exam was given. The infected subjects were clinically classified using the guidelines described by Solano-Gallego et al. (2009). The clinical diagnosis of CL was always confirmed with testing for amastigotes in lymph node and medullary aspirates, serum exam (indirect immunofluorescence - IFAT) and positive molecular test (PCR). The animals included in the exam presented clear symptoms of CL, with IFAT titres 1:80 and positive PCR results. In addition, the presence of other infective agents (Ehrlichia canis, Anaplasma phagocytophilum morulae, Babesia canis trophozoites and microfilariae) was ruled out for all of the dogs in the study by direct observation of peripheral blood smears, IFAT and/or PCR tests or rapid tests (Snap Canine Combo Heartworm Antigen Antibody Test from IDEXX) to detect Dirofilaria immitis infection. Finally, all of the subjects were carefully treated to eliminate intestinal parasites (helminthiasis) before recruitment into the study. Immunological evaluations All of the observations of the cellular phenotype and the actions of regulatory cells (phenotype and functions of the Tregs) and effector cells (T helper and cytotoxic lymphocytes) of the immune system were made using cell cultures and cytofluorimetric analysis combined with immunofluorescence with specific antibodies. Testing schedule, evaluation of efficacy The subjects were followed for a total of 12 months from the beginning of treatment. The evaluation involved: 3

4 1. Clinical evaluation - at 0 days, 30 days, 6 months and 12 months. 2. Laboratory analysis - complete blood count using an electronic Coulter counter with additional manual observation of blood smears to confirm the leukocyte count and the presence of thrombocytopaenia (exclusion of platelet aggregation in place of thrombocytopaenia), biochemical profile (nitrogen, creatinine and transaminase levels, protein electrophoretic profiles, A/G ratio) and urine exam, at 0 days, 30 days, 3 months, 6 months and 12 months. 3. Immunological analysis - (evaluation with flow cytofluorometry of CD4+, CD8+, CD4+/CD8+ ratio, interferon-gamma, interleukin-4, CD21+, CD3+) at 0 days, 30 days, 3 months, 6 months and 12 months. Statistical analysis Statistical analysis was performed using the Mann-Whitney test (GraphPad Prism, San Diego, CA, USA). Results were considered statistically significant at p<0.05. Results At the time of initial recruitment, the percent of CD8+CD3+ cytotoxic T lymphocytes was significantly higher in the diseased dogs compared to the healthy dogs. After 3 to 6 months of treatment (Group I with conventional drug therapy + Immuno Active diet, Group II with drug therapy only), the percentage of CD8+CD3+ T lymphocytes was continually elevated compared to that of the control group. It should be emphasized that the percentage of CD8+CD3+ T lymphocytes was significantly higher at 6 months in the dogs in Group I (conventional drug therapy plus Immuno Active diet). This observation was confirmed by a decrease in the CD4+/CD8+ lymphocyte ratio. The experiment also revealed a lower percentage of Treg in diseased dogs compared to healthy dogs at the time of initial recruitment. 3 to 6 months after beginning the experiment, there was an increase in the percentage of Treg in Group I (conventional drug therapy plus Immuno Active diet). The percentage of cells approached that of the healthy dogs in the control group, suggesting that the Immuno Active diet may aid recuperation. Finally, it is interesting to note that the percentage of Th1 cells (specifically producing IFN-γ and negative for IL-4) was similar and only slightly higher in infected dogs compared to healthy dogs at the time of recruitment. In contrast, after 6 months of treatment the percentage of Th1 increased 4

5 in Group I (conventional drug therapy plus Immuno Active diet) and the production of IL-4 decreased. This result also suggests that the Immuno Active diet may have immunomodulatory potential. ability to control the chronic course of the disease and the physiopathological aspects associated with the infection. Conclusions The observations made in this study suggest that the Immuno Active diet may shift the balance of the immune system of dogs infected with L. infantum, stimulating certain functions like the strengthening of cytotoxic T lymphocyte activity and T proinflammatory secretory activity, as well as the promotion of the regulation of the response itself, increasing Treg cells. As such, the increase in the cytotoxic T CD8+ population, in combination with that of the Th1 cells, may promote control of the spread of the parasite and reduce its infection load. In addition, the increase in the Treg population may correlate to a reduction in the inflammatory/immunopathogenic effects in peripheral tissues (skin and internal organs), typical of the physiopathological aspects of the disease. In conclusion, the Immuno Active diet appears to synergize with the conventional anti-leishmania therapy, providing the 5

6 The results of the above study were presented as a poster at an international conference: Abstract of the Poster presented at: WORLDLEISH 5 Porto de Galinhas, Pernabco, Brazil, May 13th to 17th, 2013 Clinical and Experimental Immunology P048 - THE EFFECT OF AN IMMUNE MODULATING DIET ON IMMUNE SYSTEM OF DOGS NATU- RALLY INFECTED BY LEISHMANIA INFANTUM - Cortese L., Annunziatella M., Piantedosi D., Palatucci A.T., Rubino V., Foglia Manzillo V., Guccione J., Ruggiero G., Oliva G. Canine leishmaniosis is a systemic parasitic disease, endemic in Mediterranean countries. It has been demonstrated that the immune system plays a key role in the development and outcome of Leishmania infection in the dog and in the response to the treatment, although this response is not well undestood. Several mechanisms account for the control of the immune response. Regulatory systems include mechanisms intrinsic to the antigen activation and to T cell differentiation, but they are also mediated by regulatory suppressor populations, as represented by the CD4+ FoxP3+ T cell subset (Treg). Our previous data (Cortese et al., 2011) showed that Leishmania infected dogs are characterised by significant increase in the percentage of cytotoxic T cell effectors (CD8+CD3+) with a decreased CD4/CD8 ratio. A significant reduction of Treg subset was also observed in infected animals. Besides, a negative correlation between Treg and CD8+CD3+ T lymphocytes was observed in dogs with leishmaniosis. The energy/metabolic status has been described to significantly modify the immune response as well as the immune tolerance control in human and animal models. The aim of this study has been to assess the impact of immune modulating diet intake on the immunological state of dogs naturally infected by L. infantum. Twenty dogs were submitted to a conventional treatment in addition to a standard feed and 20 dogs were submitted to a conventional treatment in addition to an immune modulating diet, for 1 year. Twenty healthy animals were also included in the study (control group). T lymphocytes subsets, Treg population and cytokine levels were detected by immunefluorescence and flow cytometry multi-parametric analysis. Our preliminary observations suggest as an immune modulating diet could significantly modify the immunological parameters related to the asset of effector CD8 T lymphocytes and T 6

7 regulatory cells, as well as the proinflammatory cytokine secretion. In this regard, we hypothesize that the improvement of nutritional condition obtained by a food rich in phytotherapics (with antioxidant/immunomodulant properties such as Resveratrol, Echinacea purpurea and Curcuma), with a selected sources of protein (fish hydrolysates) and optimal omega 3/6 ratio, without pharmaceutical residues in flour, could improve the immune response and the clinical outcome of L. infantum infected dogs when compared with a standard diet. References Anstead G.M., Chandrasekar B., Zhao W., Yang J., Perez L.E., Melby P.C., Malnutrition Alters the Innate Immune Response and Increases Early Visceralization following Leishmania donovani Infection. Infection and Immunity Vol. 69, No. 8, Cortese, L., Serretiello, S., Piantedosi, D., Annunziatella, M., Guccione, J., Ruggiero, G., Terrazzano, G., Ciaramella, P., Regulatory T cells are significantly decreased in dogs naturally infected by Leishmania infantum. The 21 st Annual ECVIM Congress, Sevilla, Spain. September De Lima, V.M.F., Ikeda, F.A., Rossi, C.N., Feitosa, M.M., de Oliveira Vasconcelos, R., Nunes, C.M., Goto, H., Diminished CD4 + /CD25 + T cell and increased IFN- levels occur in dogs vaccinated with Leishmune in an endemic area for visceral leishmaniosis. Veterinary Immunology and Immunopathology 135, Malafaia G., Serafim T.D., Silva M.E., Pedrosa M.L., Rezende S.A., Protein-energy malnutrition decreases immune response to Leishmania chagasi vaccine in BALB/c mice. Parasite Immunology, 2009, 31, , G., Cardoso, L., Pennisi, M.G., Ferrer, L., Bourdeau, P.,Oliva, G., Baneth, G Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Veterinary Parasitology 165,

8 University of Naples Federico II Doctor Laura Cortese (Dept. of Veterinary Medicine and Animal Production, University of Napoli FEDERICO II) Doctor Giuseppe Terrazzano (Dept. of Sciences, University of Basilicata, and Dept. of Translational Medical Sciences, University of Napoli FEDERICO II) Prof. Giuseppina Ruggiero (Dept. of Translational Medical Sciences, University of Napoli FEDERICO II) 8