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Evaluation of transformation growth factor β1, interleukin-10, and interferon-γ in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi
Veterinary Parasitology 143 (2007) 267–274 www.elsevier.com/locate/vetpar
Evaluation of transformation growth factor b1, interleukin-10, and interferon-g in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi Ana Paula Ferreira Lopes Correˆa a, Ana Cla´udia Silva Dossi a, Rosemeri de Oliveira Vasconcelos b, Danı´sio Prado Munari c, Vale´ria Marc¸al Felix de Lima b,* b
a Mestre, Microbiologia Agropecua´ria, FCAV, UNESP, Jaboticabal, S.P, Brasil Departamento de Clı´nica, Cirurgia e Reproduc¸a˜o Animal, FOA, UNESP, Arac¸atuba, S.P, Brasil c Departamento de Cieˆncia Exatas, FCAV, UNESP, Jaboticabal, S.P, Brasil
Received 29 May 2006; received in revised form 3 August 2006; accepted 3 August 2006
Abstract The aims of this study were to evaluate the immunomodulatory role of TGF-b1, IL-10, and INF-g in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Arac¸atuba, Sa˜o Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n = 15) and symptomatic (n = 15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-b1, IL-10, and INF-g. Naturally active in vitro produced TGF-b1 was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-b1 levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-g concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokine was lower in spleen extract. Although INF-g is being produced in canine infection, mean levels of TGF-b1 and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs was predominantly type Th2. # 2006 Elsevier B.V. All rights reserved. Keywords: ELISA; Visceral leishmaniasis; Spleen cell culture; Th2 response; Cytokines; Zoonosis
1. Introduction * Corresponding author at: Departamento de Clı´nica e Cirurgia e Reproduc¸a˜o Animal, Faculdade de Odontologia, UNESP, Rua Clo´vis Pestana, 793, Bairro Jardim Dona Ame´lia, CEP 16050-680, Arac¸atuba, S.P, Brasil. Tel.: +55 18 36363285; fax: +55 18 36224542. E-mail addresses: [email protected] (A.P.F.L. Correˆa), [email protected] (A.C.S. Dossi), [email protected] (R. de Oliveira Vasconcelos), [email protected] (D.P. Munari), [email protected] (V.M.F. de Lima). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.08.023
Zoonotic visceral leishmaniasis is caused by Leishmania (Leishmania) chagasi (Herwaldt, 1999). It is endemic in America, Europe, and Asian countries (Pearson and Sousa, 1996; Herwaldt, 1999) with new dissemination areas being identified (Enserink, 2000; Silva et al., 2001). Transmission between vertebrate hosts is by the phlebotomine hematophagous bite of
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Lutzomyia longipalpis (Alencar et al., 1991). Dogs (Canis familiares) were considered the most important urban reservoirs of L. (L.) chagasi (Costa and Vieira, 2001) as show a larger prevalence to infection (Paranhos-Silva et al., 1996) and from where it gets transmitted to humans (Abranches et al., 1991). Canine visceral leishmaniasis (CVL) can be considered an immunomediated disease due to the parasites capacity to modify the host’s immune system (Slappendel and Ferrer, 1990). In general, the helper CD4+ T lymphocyte population divides into two subpopulations: Type 1 (Th1) and type 2 (Th2) CD4+ T helper lymphocytes, which are defined as the basic standard cytokines (Mosmann et al., 1986). Th1 cells secrete interleukin-2 (IL-2), interleukin-12 (IL-12), and interferon-g (IFN-g) inducing, macrophage activation and cell mediated response (Cher and Mosmann, 1987). Th2 cells secrete interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), and transformation growth factor b (TGF-b), assisting antibody mediated response (Coffman et al., 1988; Bacellar et al., 2000). The initial factors which contribute to parasite inhibition or proliferation in host cells are not well understood, but cytokines such as IFN-g, IL-10, and TGF-b are known influence in Leishmania sp. replication in macrophages (Murray et al., 1995; Wilson et al., 1998; Walker et al., 1999). TGF-b is produced by many cells including B cell, T lymphocyte, NK cell, and activated macrophages (Kehrl et al., 1986; Walh, 1994). TGF-b inactivates microbicide capacity of macrophages, influencing their ability to control intracellular parasites and able to promote development of a Th2 type response (Barral et al., 1993). Leishmania sp. infection induces both in vitro and in vivo production active TGF-b in murine models, being important in its regulation (Barral-Netto et al., 1992; Barral et al., 1993). Rodrigues et al. (1998) showed in spleen cells from hamsters infected with Leishmania (Leishmania) donovani that this cytokine was abundantly produced in vivo and that high levels of TGF-b were produced in vitro by spontaneously or after antigen (parasite) or lipopolysaccharide stimulation. IL-10 is produced by activated macrophages, T cells and B cell (Howard and O’Garra, 1992; Gasim et al., 1998). IL-10 has the potential to incapacitate host defence against Leishmania sp. and promote infection visceralization and at the same time weaken responsiveness to chemotherapy in mice (Murray et al., 2002). It is the main cytokine that can negatively regulate immune response in humans with visceral leishmaniasis
and in murine models (Barral et al., 1993; Bacellar et al., 2000). INF-g is produced by T cell and NK cell (Trichieri et al., 1993). Induces synthesis of nitric oxide synthase (iNOS) and activates microbicide functions (Reiner and Locksley, 1995). Holzmuller et al. (2005) have shown that dogs immunized with L. (L.) infantum promastigota exhibit an efficient in vitro immune cell response against infection, characterized by the increase in leishmanicide activity from INF-g. INF-g limits the increase of Leishmania sp. in murine models and human macrophages and the progress of leishmaniasis. Systemic or local modulation of INF-g levels can be a critical determinant in resolving the disease (Murray et al., 1992). Santos-Gomes et al. (2002), observed that the proportion of non-stimulated or specifically stimulated infected dogs expressing IL-12, IL-2, and INF-g, was generally very low for a long time after infection (8 months) and this, associated with low IL-10 detection, suggests the parasite has ‘‘established silence’’ and too that the low proportion of dogs expressing IL-10, it may not have a direct inhibitory effect on INF-g. IL-10 and INF-g coexist in cutaneous or visceral human leishmaniasis, as they have both been detected. The objective of this study is to evaluate the presence of cytokines related to Th1 profile: INF-g and Th2 profile: TGF-b1 and IL-10, in male dogs naturally infected by L. (L.) chagasi, with or without clinical manifestation, to improve understanding of immunological modulation and response to the parasite, and also to develop treatment and prevent this condition. 2. Materials and methods 2.1. Study area The study was conducted in Arac¸atuba, Sa˜o Paulo State, Brazil, an endemic area for canine visceral leishmaniasis. 2.2. Animals Thirty adult male dogs between 2 and 5 years old, of various breeds and weights, were provided from the Arac¸atuba Zoonosis Control Centre (ZCC); they were seropositive for L. (L.) chagasi, tested by indirect ELISA as per Lima et al. (2003). They were subdivided into two groups: asymptomatic (n = 15), and symptomatic (n = 15) consisting of animals with at least three characteristic clinical signs
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(fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy).
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Animals were despatched by the following process: prior tranquilizing with 0.05 mg/kg acepromazine maleate IV; 15 min interval; quick injection (20 s) of 15 mg/kg sodium thiopental IV followed by potassium chlorate (10 ml ampoule) by the same route. Samples were obtained of total blood, and spleen and liver fragments which were used for active TGF-b1, and IL10 and INF-g evaluation. Total blood was placed in a refrigerator and organ fragments were immediately preserved in liquid nitrogen to avoid cytokine degradation.
with L. (L.) chagasi. As active TGF-b1 was found in serum, samples of human and dog (male and female) serum were separated by 10% SDS-PAGE and electrotransferred (Biorad, USA) to nitrocellulose membrane. The membrane was buffered with PBS (0.01 M, pH 7.2), added to 5% skimmed milk, incubated for 2 h at ambient temperature. After washing with PBS (0.01 M, pH 7.2), the membrane was incubated for 2 h with TGF-b1 monoclonal antibody diluted in buffer solution (1:1000). After five washes for 10 min, it was incubated for another 2 h with biotin conjugated rat polyclonal anti-IgG (PharMigen, USA) (1:500). The reaction was amplified with streptoavidin– biotin peroxidase (DAKO Corporation, Denmark) (1:150) for 45 min. After another five washes, the membrane was developed with a solution of PBS (0.01 M, pH 7.2) containing 1 mg/ml diaminobenzedine (DAB) and 30 ml hydrogen peroxide (H2O2).
2.4. White spleen cell culture
2.7. TGF-b1 quantification
White spleen cells were cultured to obtain naturally produced active TGF-b1 in vitro. Spleen was pulverized in complete RPMI-1640 medium (Sigma, USA), pH 7.2. After lysis the resulting pellet was washed three times with sterile PBS and suspended in 1 ml of complete RPMI-1640 medium. White cells were plated at 5 106 cells/well on 24 well plates (TPT-92024, Switzerland). After incubation for 24 h at 37 8C in a humidified incubator under 5% CO2, supernatant was collected, centrifuged, and stored at 80 8C (Revco, USA). The complete procedure was performed under sterile laminar flow conditions.
Active TGF-b1 quantification was made from spleen and liver extract and also from 24 h supernatant from white spleen cells. Evaluation was by ELISA sandwich test, using a commercial human kit (Promega Corporation, USA) as per manufacturer’s instructions. Plates were read by a Spectra CountTM reader (Packard Bio Science Company, USA), with 490 nm filter. Minimum sensitivity was 580 pg/ml.
2.3. Material sampling
2.5. Spleen and liver extract Extracts were obtained to quantify TGF-b1, IL-10, and IFN-g cytokines by the ELISA sandwich technique. For this, 1 g spleen and liver fragments and 2 ml suspensions of complete RPMI-1640 (Sigma, USA), pH 7.2, were kept in ice and ground in a tissue homogenizer (Ultraturrax T8, Germany) for approximately 5 min. The resulting homogenate was centrifuged at 10,000 g for 15 min at 4 8C and the supernatant was immediately stored at 80 8C (Revco, USA).
2.8. IL-10 and INF-g quantification IL-10 and INF-g quantification used spleen and liver extracts and ELISA sandwich with anticanine monoclonal antibody (mAb) produced in mice and biotinated anticanine polyclonal antibody produced in goat (R&D Systems, USA). Plates with 96 wells (Nalgene Nunc International, USA) were sensitized with 0.5 mg/ml mAb and 0.125 mg/ml detection antibody, respectively. Recombinant canine INF-g and IL-10 (R&D Systems) were used to generate standard curves. The test was developed with 3,30 ,5,50 -tetramethylbenzedine—TMB (Promega Corporation) in accordance with manufacturer’s instructions, and plates were red by Spectra CountTM reader (Packard Bio Science Company) with 450 nm filter. Minimum sensitivity was 380 pg/ml for IL-10 and 9.6 pg/ml for INF-g.
2.6. Western blot 2.9. Statistical analysis This technique was used to validate the use of the commercial human ELISA active TGF-b1 kit (Promega Corporation, USA) for use in dogs naturally infected
Measurements from each group (asymptomatic and symptomatic), for each variable (TGF-b in spleen, liver,
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and culture; IL-10 in spleen and liver; and INF-g in spleen and liver) were made in triplicate and means taken. These means were submitted to analysis of variance for a general linear model including fixed status effect at two levels (asymptomatic and symptomatic). To address basic test presuppositions (residue normality and homocedasticity), values less than zero were ignored (for TGF-b from culture, and spleen and liver INF-g), and spleen and liver TGF-b and IL-10, and spleen INF-g were submitted to logarithm transforms, and liver INF-g to square root. Significance was set at 5%. Statistical analyses were made using the SAS program (Statistical Analysis System, SAS Institute) and graphs were developed in Microsoft Excel 2000. 3. Results 3.1. TGF-b1 quantification The Western blot test indicated crossed reactivity between human antibodies and those found in dog serum samples (Fig. 1) and quantifying from this procedure, we observed mean TGF-b1 production in spleen and liver extract from both dog groups. There were significant differences between asymptomatic and symptomatic dogs in both organ extracts (P < 0.05). Asymptomatic dogs had mean spleen and liver TGF-b1 values 1.5 times higher than symptomatic dogs (Fig. 2A and B, respectively). Spleen mean TGF-b1 production was three times higher than in the liver in both asymptomatic and symptomatic dog groups. TGF-b1 was detected in spleen white cell culture (Fig. 2C). Symptomatic group mean production was double the asymptomatic group, this was significantly different.
Fig. 2. TGF-b1 quantification by ELISA, on spleen (A) and liver (B) extract, and spleen white cells culture supernatant (C) from asymptomatic and symptomatic male dogs naturally infected by L. (L.) chagasi. Different letters indicate significant differences (P < 0.05) between means of the two groups by analysis of variance. Asymptomatic dogs had higher TGF-b1 production ex vivo and symptomatic dogs had higher TGF-b1 production in vitro.
Ex vivo results showed higher mean TGF-b1 production in asymptomatic dogs whereas in vitro results showed higher production in symptomatic dogs. 3.2. IL-10 quantification
Fig. 1. Bands formed by the Western blot technique showing cross relativity between the species (1: human serum sample; 2: female dog serum sample; 3: male dog serum sample).
IL-10 production was seen in spleen and liver extracts (Fig. 3A and B). High levels were recorded but mean values were not significantly different (P < 0.05). Mean IL-10 production was five times higher in liver extract than in spleen extract from asymptomatic dogs; symptomatic dogs also had higher mean IL-10 production values in liver extract.
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Fig. 3. IL-10 quantification by ELISA, on spleen (A) and liver (B) extract from asymptomatic and symptomatic male dogs naturally infected by L. (L.) chagasi. The same letter indicates that no significant differences were found (P < 0.05) between means of the two groups by analysis of variance. Overproduction of IL-10 was seen mainly in liver extract.
3.3. INF-g quantification INF-g production was observed in spleen and liver extracts. There were significant differences between groups for both organ extracts. Symptomatic group spleen extract (Fig. 4A) was 5.5 times higher than in asymptomatic dogs. Asymptomatic group liver extract (Fig. 4B) was 2.4 times higher than in symptomatic dogs. Comparing mean INF-g production between organs, asymptomatic mean liver concentration was 230 times higher than in the spleen, and in symptomatic dogs, mean liver concentration was 18 times higher than in the spleen. In brief, there was higher mean INF-g production in liver extract. 4. Discussion We studied 30 dogs naturally infected by L. (L.) chagasi from Arac¸atuba, Sa˜o Paulo, Brazil. They were divided into an asymptomatic and a symptomatic group.
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Fig. 4. INF-g quantification by ELISA, on spleen (A) and liver (B) extract from asymptomatic and symptomatic male dogs naturally infected by L. (L.) chagasi. Different letters indicate significant differences (P < 0.05) between means of the two groups by analysis of variance. In general, spleen extract had low mean levels of INF-g production. Symptomatic dogs had higher levels in spleen extract then the other group, and asymptomatic dogs had higher levels in the liver than the other group.
Western blot results confirmed crossed reactivity between active human and active canine anti-TGF-b1 monoclonal antibody as observed by Vercelli et al. (2003). This allowed us to evaluate mean TGF-b1 production in our study groups. TGF-b1 is an important regulator cytokine in CVL. It has been associated with the disease’s progress in murine models (Bacellar et al., 2000). There are no studies on the immunoregulatory role of TGF-b1 to visceral leishmaniasis in dogs. Results from our study showed this cytokine produced in both groups of dogs (asymptomatic and symptomatic), and ex vivo and in vitro (Fig. 2), suggesting that TGF-b1 was actually induced by the Leishmania sp. infection as confirmed by Barral-Netto et al. (1992) and Barral et al. (1993) in murine models. Barral-Netto et al. (1992), Bright and Sriram (1998) and Wilson et al. (1998) observed that, among other things, TGF-b is capable of inhibiting the signal that initiates INF-g production; also Rodrigues et al. (1998) saw high TGF-b1 levels produced by the spleen in vitro and ex vivo. Our results showed elevated mean TGF-b1
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and low INF-g production in spleen extract suggesting that TGF-b1 inhibits INF-g production. We observed that the spleen is a large TGF-b1 producer, a fact that could be associated with the non-protector immunological response in dogs after infection. Barral et al. (1993) showed that TGF-b1 also suppresses macrophage microbicide mechanisms, having the power to promote a Th2 type immune response. Our results agree with this as we found high levels of both TGF-b1 and IL-10 which promote this type of humeral immune response and are not linked to disease resolution. Ex vivo IL-10 production was found in spleen and liver extracts (Fig. 3A and B) of asymptomatic and symptomatic dogs. It is the main cytokine that suppresses protector immune response in murine models and humans with visceral leishmaniosis (VL) (Barral et al., 1993; Bacellar et al., 2000). Mean IL-10 production was higher than the other cytokines studied; this was more pronounced in liver extract. Bogdan et al. (1991), Karp et al. (1993), Wilson et al. (1998), and Bacellar et al. (2000) confirmed that this cytokine is very active in various T cell mechanisms and is also capable of inhibiting the secretion of cytokines which active Th1 cells. Our results clearly demonstrate elevated INF-g in liver extract of both dog groups, but five times lower than IL-10 in this organ; this suggests that IL-10 production in the liver is inhibiting INF-g making it the main regulatory cytokine in CVL. This elevated IL-10 production in asymptomatic and symptomatic dogs is similar to that seen by Ghalib et al. (1993), suggesting that the immune response in dogs is similar to that in human patients with acute VL. The high IL-10 production seen indicates that macrophages and T cells from infected dogs are producing IL-10 in response to the leishmania antigen and that this production could be important in the persistence of the parasite in host cells. Observation of raw IL-10 production data analysis shows a tendency towards higher production in symptomatic dogs, confirming studies in humans where disease severity and tissue lesion are associated with high serum levels of IL-10 (Gasim et al., 1998; Ghalib et al., 1993). Similarities observed in immunological response between naturally infected dogs and patients with acute VL suggest that the dog is an excellent model to study new therapies. In dogs inoculated with L. (L.) infantum amastigotes, Santos-Gomes et al. (2002) observed low proportion of animals which express IL-10 and suggested that this may not have a direct inhibitor effect on INF-g. Our
results were different; all studied dogs produced IL-10, and it is important to highlight the difference between natural and experimental infection, and that possibly, the initial immunomodulatory role of saliva on bleeding could play a fundamental in subsequent adaptive response (Paranhos et al., 1993). Our results indicate the coexistence of IL-10 and IFN-g in both dog groups naturally infected by L. (L.) chagasi similar to that seen in human VL (Ansari et al., 2006). The elevated IL-10 production could cause an alteration in macrophage activity, which could increase parasitemia and disease progress. INF-g, the cytokine related to VL resolution in humans and murine models (Wilson et al., 1998), produced less than the other cytokines studied. Our spleen extract results showed that symptomatic dogs produce higher quantities than the asymptomatic group. Disease chronicity in the dog seems to be associated to increased spleen IFN-g. The observation that high IFNg levels seem to be associated with worse disease prognosis in the dog indicates a failure in response to this cytokine, which is similar to a study on humans where the disease was prolonged by a lack of response to treatment (Ansari et al., 2006). Interestingly, that reduced expression of IFN-g R1 receptor in humans with VL has already been reported (Dasgupta et al., 2003) but there has been no study in dogs. Quinnell et al. (2001) using RT-PCR, considered that independently analysed levels of INF-g may not be good indicators of disease resistance, because similar levels of INF-g were found in tissue of both asymptomatic and symptomatic dogs. Our results were similar with this study, because INF-g production was seen in both groups of dogs. In dogs, isolated analysis of INF-g is not a good indicator of CVL status or eventual cure; this is similar to that suggested by Karp et al. (1993). Few studies have shown the profile of cytokines in dogs infected by L. (L.) chagasi; in this quantitative study we saw that asymptomatic dogs generally produced lower mean quantities of cytokines than symptomatic dogs; seen by the higher mean production levels of TGF-b1 and IL-10, supporting the observation of a predominance of type Th2 immune response, similar to that suggested by Santos-Gomes et al. (2002). Santos-Gomes et al. (2002) observed that Th1 response was capable of slowing down the start of the disease, but incapable of controlling it, and Chamizo et al. (2005) also observed that although both cytokine profiles are produced in asymptomatic dogs infected by Leishmania sp., only Th1 response gives immunity against the parasite. The results of this study
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demonstrated the production of Th1 cytokine (INF-g) in both asymptomatic and symptomatic dogs and in both organs, but it was the predominance of Th2 cytokines (TGF-b1 and IL-10), as shown by Quinnell et al. (2001), that these determine the disease progress and symptom appearance. In summary, there was a higher mean production of TGF-b1 in the spleen, a higher mean production of IL-10 in the liver, and lower production of IFN-g in both organs; this suggests that cytokines related to Th2 profile, IL-10 and TGF-b1 may be inhibiting their production. Both asymptomatic and symptomatic dogs produce cytokines belonging to both Th1 and Th2 profiles, and that these coexist in CVL. In conclusion, our findings show that dogs naturally infected by L. (L.) chagasi, have a strong ability to develop a type Th2 response. Acknowledgements To FAPESP and CAPES for financially supporting this project. References Abranches, P., Silva-Pereira, M.C., Conceic¸a˜o-Silva, F.M., SantosGomes, G.M., Janz, J.G., 1991. Canine leishmaniasis: pathological and ecological factors influencing transmission of infection. J. Parasitol. 77, 557–561. Alencar, J.E., Neves, J., Dietze, R., 1991. Leishmaniose visceral (Calazar). In: Veronezi, R. (Ed.), Doenc¸as Infecciosas e Parasita´rias. eighth ed. Guanabara Koogan, Rio de Janeiro, pp. 706–717. Ansari, N.A., Saluja, S., Salotra, P., 2006. Elevated levels of interferon-gamma, interleukin-10, and interleukin-6 during active disease in Indian kala azar. Clin. Immunol. 119, 339–345. Bacellar, O., D’Oliveira Jr., A., Jeroˆnimo, S., Carvalho, E.M., 2000. IL-10 and IL-12 are the main regulatory cytokines in visceral leishmaniasis. Cytokine 12, 1228–1231. Barral, A., Barral-Netto, M., Yong, E.C., Brownell, C.E., Twardzik, D.R., Reed, S.G., 1993. Transforming growth factor b as a virulence mechanism for Leishmania braziliensis. Proc. Natl. Acad. Sci. U.S.A. 90, 3442–3446. Barral-Netto, M., Barral, A., Brownell, C.E., Skeiky, Y.A.W., Ellingsworth, L.R., Twardzik, D.R., Reed, S.G., 1992. Transforming growth factor-b in leishmanial infection: a parasite escape mechanism. Science 257, 545–548. Bogdan, C., Vodovotz, Y., Nathan, C., 1991. Macrophage deactivation by interleukin 10. J. Exp. Med. 174, 1549–1555. Bright, J.J., Sriram, S., 1998. TGF-b inhibits IL-12-induced activation of Jak-STAT pathway in T lymphocytes. J. Immunol. 161, 1772– 1777. Chamizo, C., Moreno, J., Alvar, J., 2005. Semi-quantitative analysis of cytokine expression in asymptomatic canine leishmaniasis. Vet. Immunol. Immunopathol. 103, 67–75. Cher, D.J., Mosmann, T.R., 1987. Two types of murine helper cell clone. II Delayed-type hypersensitivity is mediated by Th1 clones. J. Immunol. 138, 3688–3694.
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Paranhos, M., dos Santos, W.C., Sherlock, I., Oliveira, G.G., de Carvalho, L.C., 1993. Development of eosinophilia in dogs intradermically inoculated with sand fly saliva and Leishmania (Leishmania) chagasi stationary-phase promastigotes. Mem. Inst. Oswaldo Cruz 88, 249–251. Paranhos-Silva, M., Freitas, L.A., Santos, W.C., Grimaldi Jr., G., Pontes-de-Carvalho, L.C., Oliveira-dos-Santos, A.J., 1996. A cross-sectional serodiagnostic survey of canine leishmaniasis due to Leishmania chagasi. Am. J. Trop. Med. Hyg. 55, 39–44. Pearson, R.D., Sousa, A.Q., 1996. Clinical spectrum of leishmaniasis. Clin. Infect. Dis. 22, 1–13. Quinnell, R.J., Courtenay, O., Shaw, M.A., Day, M.J., Garcez, L.M., Dye, C., Kaye, P.M., 2001. Tissue cytokine responses in canine visceral leishmaniasis. J. Infect. Dis. 183, 1421–1424. Reiner, S.L., Locksley, R.M., 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13, 151–177. RodriguesFV. Jr., Silva, J.S., Campos-Neto, A., 1998. Transforming growth factor b and immunosupression in experimental visceral leishmaniasis. Infect. Immun. 66, 1233–1236. Santos-Gomes, G.M., Rosa, R., Leandro, C., Cortes, S., Romao, P., Silveira, H., 2002. Cytokine expression during the outcome of canine experimental infection by Leishmania infantum. Vet. Immunol. Immunopathol. 88, 21–30. Silva, E.S., Gontijo, C.M., Pacheco, R.S., Fiu´za, V.O., Brazil, R.P., 2001. Visceral leishmaniasis in Metropolitan region of Belo
Horizonte, State of Minas Gerais, Brazil. Mem. Inst. Oswaldo Cruz 96, 285–291. Slappendel, R.J., Ferrer, L., 1990. Leishmaniasis. In: Greene, C.E. (Ed.), Clinical Microbiology and Infectious Diseases of the Dog and Cat. W.B. Saunders Co., Philadelphia, pp. 450–458. Trichieri, G., Rengaraju, M., D’Andrea, A., Valiante, N.M., Kubin, M., Aste, M., Chehimi, J., 1993. Producer cells of interleukin-12. Immunol. Today 14, 237–238. Vercelli, A., Bellone, G., Abate, O., Emanuelli, G., Cagnasso, A., 2003. Expression of transforming growth factor b isoforms in the skin, kidney, pancreas and bladder in german shepherd dog affected by renal cystadenocarcinoma and nodular dermatofibrosis. J. Vet. Med. 50, 506–510. Walh, S.M., 1994. Transforming growth factor beta: the good, the bad, and the ugly. J. Exp. Med. 180, 1587–1590. Walker, P.S., Scharton-Kersten, T., Krieg, A.M., Love-Homan, L., Rowton, E.D., Udey, M.C., Vogel, J.C., 1999. Immunoestimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12 and IFN-g dependent mechanisms. Proc. Natl. Acad. Sci. U.S.A. 96, 6970–6975. Wilson, M.E., Young, B.M., Davidson, B.L., Mente, K.A., McGowan, S.E., 1998. The importance of transforming growth factor b in murine visceral leishmaniasis. J. Immunol. 161, 6148–6155.
Evaluation of transformation growth factor b1, interleukin-10, and interferon-g in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi Ana Paula Ferreira Lopes Correˆa a, Ana Cla´udia Silva Dossi a, Rosemeri de Oliveira Vasconcelos b, Danı´sio Prado Munari c, Vale´ria Marc¸al Felix de Lima b,* b
a Mestre, Microbiologia Agropecua´ria, FCAV, UNESP, Jaboticabal, S.P, Brasil Departamento de Clı´nica, Cirurgia e Reproduc¸a˜o Animal, FOA, UNESP, Arac¸atuba, S.P, Brasil c Departamento de Cieˆncia Exatas, FCAV, UNESP, Jaboticabal, S.P, Brasil
Received 29 May 2006; received in revised form 3 August 2006; accepted 3 August 2006
Abstract The aims of this study were to evaluate the immunomodulatory role of TGF-b1, IL-10, and INF-g in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Arac¸atuba, Sa˜o Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n = 15) and symptomatic (n = 15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-b1, IL-10, and INF-g. Naturally active in vitro produced TGF-b1 was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-b1 levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-g concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokine was lower in spleen extract. Although INF-g is being produced in canine infection, mean levels of TGF-b1 and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs was predominantly type Th2. # 2006 Elsevier B.V. All rights reserved. Keywords: ELISA; Visceral leishmaniasis; Spleen cell culture; Th2 response; Cytokines; Zoonosis
1. Introduction * Corresponding author at: Departamento de Clı´nica e Cirurgia e Reproduc¸a˜o Animal, Faculdade de Odontologia, UNESP, Rua Clo´vis Pestana, 793, Bairro Jardim Dona Ame´lia, CEP 16050-680, Arac¸atuba, S.P, Brasil. Tel.: +55 18 36363285; fax: +55 18 36224542. E-mail addresses: [email protected] (A.P.F.L. Correˆa), [email protected] (A.C.S. Dossi), [email protected] (R. de Oliveira Vasconcelos), [email protected] (D.P. Munari), [email protected] (V.M.F. de Lima). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.08.023
Zoonotic visceral leishmaniasis is caused by Leishmania (Leishmania) chagasi (Herwaldt, 1999). It is endemic in America, Europe, and Asian countries (Pearson and Sousa, 1996; Herwaldt, 1999) with new dissemination areas being identified (Enserink, 2000; Silva et al., 2001). Transmission between vertebrate hosts is by the phlebotomine hematophagous bite of
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Lutzomyia longipalpis (Alencar et al., 1991). Dogs (Canis familiares) were considered the most important urban reservoirs of L. (L.) chagasi (Costa and Vieira, 2001) as show a larger prevalence to infection (Paranhos-Silva et al., 1996) and from where it gets transmitted to humans (Abranches et al., 1991). Canine visceral leishmaniasis (CVL) can be considered an immunomediated disease due to the parasites capacity to modify the host’s immune system (Slappendel and Ferrer, 1990). In general, the helper CD4+ T lymphocyte population divides into two subpopulations: Type 1 (Th1) and type 2 (Th2) CD4+ T helper lymphocytes, which are defined as the basic standard cytokines (Mosmann et al., 1986). Th1 cells secrete interleukin-2 (IL-2), interleukin-12 (IL-12), and interferon-g (IFN-g) inducing, macrophage activation and cell mediated response (Cher and Mosmann, 1987). Th2 cells secrete interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), and transformation growth factor b (TGF-b), assisting antibody mediated response (Coffman et al., 1988; Bacellar et al., 2000). The initial factors which contribute to parasite inhibition or proliferation in host cells are not well understood, but cytokines such as IFN-g, IL-10, and TGF-b are known influence in Leishmania sp. replication in macrophages (Murray et al., 1995; Wilson et al., 1998; Walker et al., 1999). TGF-b is produced by many cells including B cell, T lymphocyte, NK cell, and activated macrophages (Kehrl et al., 1986; Walh, 1994). TGF-b inactivates microbicide capacity of macrophages, influencing their ability to control intracellular parasites and able to promote development of a Th2 type response (Barral et al., 1993). Leishmania sp. infection induces both in vitro and in vivo production active TGF-b in murine models, being important in its regulation (Barral-Netto et al., 1992; Barral et al., 1993). Rodrigues et al. (1998) showed in spleen cells from hamsters infected with Leishmania (Leishmania) donovani that this cytokine was abundantly produced in vivo and that high levels of TGF-b were produced in vitro by spontaneously or after antigen (parasite) or lipopolysaccharide stimulation. IL-10 is produced by activated macrophages, T cells and B cell (Howard and O’Garra, 1992; Gasim et al., 1998). IL-10 has the potential to incapacitate host defence against Leishmania sp. and promote infection visceralization and at the same time weaken responsiveness to chemotherapy in mice (Murray et al., 2002). It is the main cytokine that can negatively regulate immune response in humans with visceral leishmaniasis
and in murine models (Barral et al., 1993; Bacellar et al., 2000). INF-g is produced by T cell and NK cell (Trichieri et al., 1993). Induces synthesis of nitric oxide synthase (iNOS) and activates microbicide functions (Reiner and Locksley, 1995). Holzmuller et al. (2005) have shown that dogs immunized with L. (L.) infantum promastigota exhibit an efficient in vitro immune cell response against infection, characterized by the increase in leishmanicide activity from INF-g. INF-g limits the increase of Leishmania sp. in murine models and human macrophages and the progress of leishmaniasis. Systemic or local modulation of INF-g levels can be a critical determinant in resolving the disease (Murray et al., 1992). Santos-Gomes et al. (2002), observed that the proportion of non-stimulated or specifically stimulated infected dogs expressing IL-12, IL-2, and INF-g, was generally very low for a long time after infection (8 months) and this, associated with low IL-10 detection, suggests the parasite has ‘‘established silence’’ and too that the low proportion of dogs expressing IL-10, it may not have a direct inhibitory effect on INF-g. IL-10 and INF-g coexist in cutaneous or visceral human leishmaniasis, as they have both been detected. The objective of this study is to evaluate the presence of cytokines related to Th1 profile: INF-g and Th2 profile: TGF-b1 and IL-10, in male dogs naturally infected by L. (L.) chagasi, with or without clinical manifestation, to improve understanding of immunological modulation and response to the parasite, and also to develop treatment and prevent this condition. 2. Materials and methods 2.1. Study area The study was conducted in Arac¸atuba, Sa˜o Paulo State, Brazil, an endemic area for canine visceral leishmaniasis. 2.2. Animals Thirty adult male dogs between 2 and 5 years old, of various breeds and weights, were provided from the Arac¸atuba Zoonosis Control Centre (ZCC); they were seropositive for L. (L.) chagasi, tested by indirect ELISA as per Lima et al. (2003). They were subdivided into two groups: asymptomatic (n = 15), and symptomatic (n = 15) consisting of animals with at least three characteristic clinical signs
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(fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy).
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Animals were despatched by the following process: prior tranquilizing with 0.05 mg/kg acepromazine maleate IV; 15 min interval; quick injection (20 s) of 15 mg/kg sodium thiopental IV followed by potassium chlorate (10 ml ampoule) by the same route. Samples were obtained of total blood, and spleen and liver fragments which were used for active TGF-b1, and IL10 and INF-g evaluation. Total blood was placed in a refrigerator and organ fragments were immediately preserved in liquid nitrogen to avoid cytokine degradation.
with L. (L.) chagasi. As active TGF-b1 was found in serum, samples of human and dog (male and female) serum were separated by 10% SDS-PAGE and electrotransferred (Biorad, USA) to nitrocellulose membrane. The membrane was buffered with PBS (0.01 M, pH 7.2), added to 5% skimmed milk, incubated for 2 h at ambient temperature. After washing with PBS (0.01 M, pH 7.2), the membrane was incubated for 2 h with TGF-b1 monoclonal antibody diluted in buffer solution (1:1000). After five washes for 10 min, it was incubated for another 2 h with biotin conjugated rat polyclonal anti-IgG (PharMigen, USA) (1:500). The reaction was amplified with streptoavidin– biotin peroxidase (DAKO Corporation, Denmark) (1:150) for 45 min. After another five washes, the membrane was developed with a solution of PBS (0.01 M, pH 7.2) containing 1 mg/ml diaminobenzedine (DAB) and 30 ml hydrogen peroxide (H2O2).
2.4. White spleen cell culture
2.7. TGF-b1 quantification
White spleen cells were cultured to obtain naturally produced active TGF-b1 in vitro. Spleen was pulverized in complete RPMI-1640 medium (Sigma, USA), pH 7.2. After lysis the resulting pellet was washed three times with sterile PBS and suspended in 1 ml of complete RPMI-1640 medium. White cells were plated at 5 106 cells/well on 24 well plates (TPT-92024, Switzerland). After incubation for 24 h at 37 8C in a humidified incubator under 5% CO2, supernatant was collected, centrifuged, and stored at 80 8C (Revco, USA). The complete procedure was performed under sterile laminar flow conditions.
Active TGF-b1 quantification was made from spleen and liver extract and also from 24 h supernatant from white spleen cells. Evaluation was by ELISA sandwich test, using a commercial human kit (Promega Corporation, USA) as per manufacturer’s instructions. Plates were read by a Spectra CountTM reader (Packard Bio Science Company, USA), with 490 nm filter. Minimum sensitivity was 580 pg/ml.
2.3. Material sampling
2.5. Spleen and liver extract Extracts were obtained to quantify TGF-b1, IL-10, and IFN-g cytokines by the ELISA sandwich technique. For this, 1 g spleen and liver fragments and 2 ml suspensions of complete RPMI-1640 (Sigma, USA), pH 7.2, were kept in ice and ground in a tissue homogenizer (Ultraturrax T8, Germany) for approximately 5 min. The resulting homogenate was centrifuged at 10,000 g for 15 min at 4 8C and the supernatant was immediately stored at 80 8C (Revco, USA).
2.8. IL-10 and INF-g quantification IL-10 and INF-g quantification used spleen and liver extracts and ELISA sandwich with anticanine monoclonal antibody (mAb) produced in mice and biotinated anticanine polyclonal antibody produced in goat (R&D Systems, USA). Plates with 96 wells (Nalgene Nunc International, USA) were sensitized with 0.5 mg/ml mAb and 0.125 mg/ml detection antibody, respectively. Recombinant canine INF-g and IL-10 (R&D Systems) were used to generate standard curves. The test was developed with 3,30 ,5,50 -tetramethylbenzedine—TMB (Promega Corporation) in accordance with manufacturer’s instructions, and plates were red by Spectra CountTM reader (Packard Bio Science Company) with 450 nm filter. Minimum sensitivity was 380 pg/ml for IL-10 and 9.6 pg/ml for INF-g.
2.6. Western blot 2.9. Statistical analysis This technique was used to validate the use of the commercial human ELISA active TGF-b1 kit (Promega Corporation, USA) for use in dogs naturally infected
Measurements from each group (asymptomatic and symptomatic), for each variable (TGF-b in spleen, liver,
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and culture; IL-10 in spleen and liver; and INF-g in spleen and liver) were made in triplicate and means taken. These means were submitted to analysis of variance for a general linear model including fixed status effect at two levels (asymptomatic and symptomatic). To address basic test presuppositions (residue normality and homocedasticity), values less than zero were ignored (for TGF-b from culture, and spleen and liver INF-g), and spleen and liver TGF-b and IL-10, and spleen INF-g were submitted to logarithm transforms, and liver INF-g to square root. Significance was set at 5%. Statistical analyses were made using the SAS program (Statistical Analysis System, SAS Institute) and graphs were developed in Microsoft Excel 2000. 3. Results 3.1. TGF-b1 quantification The Western blot test indicated crossed reactivity between human antibodies and those found in dog serum samples (Fig. 1) and quantifying from this procedure, we observed mean TGF-b1 production in spleen and liver extract from both dog groups. There were significant differences between asymptomatic and symptomatic dogs in both organ extracts (P < 0.05). Asymptomatic dogs had mean spleen and liver TGF-b1 values 1.5 times higher than symptomatic dogs (Fig. 2A and B, respectively). Spleen mean TGF-b1 production was three times higher than in the liver in both asymptomatic and symptomatic dog groups. TGF-b1 was detected in spleen white cell culture (Fig. 2C). Symptomatic group mean production was double the asymptomatic group, this was significantly different.
Fig. 2. TGF-b1 quantification by ELISA, on spleen (A) and liver (B) extract, and spleen white cells culture supernatant (C) from asymptomatic and symptomatic male dogs naturally infected by L. (L.) chagasi. Different letters indicate significant differences (P < 0.05) between means of the two groups by analysis of variance. Asymptomatic dogs had higher TGF-b1 production ex vivo and symptomatic dogs had higher TGF-b1 production in vitro.
Ex vivo results showed higher mean TGF-b1 production in asymptomatic dogs whereas in vitro results showed higher production in symptomatic dogs. 3.2. IL-10 quantification
Fig. 1. Bands formed by the Western blot technique showing cross relativity between the species (1: human serum sample; 2: female dog serum sample; 3: male dog serum sample).
IL-10 production was seen in spleen and liver extracts (Fig. 3A and B). High levels were recorded but mean values were not significantly different (P < 0.05). Mean IL-10 production was five times higher in liver extract than in spleen extract from asymptomatic dogs; symptomatic dogs also had higher mean IL-10 production values in liver extract.
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Fig. 3. IL-10 quantification by ELISA, on spleen (A) and liver (B) extract from asymptomatic and symptomatic male dogs naturally infected by L. (L.) chagasi. The same letter indicates that no significant differences were found (P < 0.05) between means of the two groups by analysis of variance. Overproduction of IL-10 was seen mainly in liver extract.
3.3. INF-g quantification INF-g production was observed in spleen and liver extracts. There were significant differences between groups for both organ extracts. Symptomatic group spleen extract (Fig. 4A) was 5.5 times higher than in asymptomatic dogs. Asymptomatic group liver extract (Fig. 4B) was 2.4 times higher than in symptomatic dogs. Comparing mean INF-g production between organs, asymptomatic mean liver concentration was 230 times higher than in the spleen, and in symptomatic dogs, mean liver concentration was 18 times higher than in the spleen. In brief, there was higher mean INF-g production in liver extract. 4. Discussion We studied 30 dogs naturally infected by L. (L.) chagasi from Arac¸atuba, Sa˜o Paulo, Brazil. They were divided into an asymptomatic and a symptomatic group.
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Fig. 4. INF-g quantification by ELISA, on spleen (A) and liver (B) extract from asymptomatic and symptomatic male dogs naturally infected by L. (L.) chagasi. Different letters indicate significant differences (P < 0.05) between means of the two groups by analysis of variance. In general, spleen extract had low mean levels of INF-g production. Symptomatic dogs had higher levels in spleen extract then the other group, and asymptomatic dogs had higher levels in the liver than the other group.
Western blot results confirmed crossed reactivity between active human and active canine anti-TGF-b1 monoclonal antibody as observed by Vercelli et al. (2003). This allowed us to evaluate mean TGF-b1 production in our study groups. TGF-b1 is an important regulator cytokine in CVL. It has been associated with the disease’s progress in murine models (Bacellar et al., 2000). There are no studies on the immunoregulatory role of TGF-b1 to visceral leishmaniasis in dogs. Results from our study showed this cytokine produced in both groups of dogs (asymptomatic and symptomatic), and ex vivo and in vitro (Fig. 2), suggesting that TGF-b1 was actually induced by the Leishmania sp. infection as confirmed by Barral-Netto et al. (1992) and Barral et al. (1993) in murine models. Barral-Netto et al. (1992), Bright and Sriram (1998) and Wilson et al. (1998) observed that, among other things, TGF-b is capable of inhibiting the signal that initiates INF-g production; also Rodrigues et al. (1998) saw high TGF-b1 levels produced by the spleen in vitro and ex vivo. Our results showed elevated mean TGF-b1
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and low INF-g production in spleen extract suggesting that TGF-b1 inhibits INF-g production. We observed that the spleen is a large TGF-b1 producer, a fact that could be associated with the non-protector immunological response in dogs after infection. Barral et al. (1993) showed that TGF-b1 also suppresses macrophage microbicide mechanisms, having the power to promote a Th2 type immune response. Our results agree with this as we found high levels of both TGF-b1 and IL-10 which promote this type of humeral immune response and are not linked to disease resolution. Ex vivo IL-10 production was found in spleen and liver extracts (Fig. 3A and B) of asymptomatic and symptomatic dogs. It is the main cytokine that suppresses protector immune response in murine models and humans with visceral leishmaniosis (VL) (Barral et al., 1993; Bacellar et al., 2000). Mean IL-10 production was higher than the other cytokines studied; this was more pronounced in liver extract. Bogdan et al. (1991), Karp et al. (1993), Wilson et al. (1998), and Bacellar et al. (2000) confirmed that this cytokine is very active in various T cell mechanisms and is also capable of inhibiting the secretion of cytokines which active Th1 cells. Our results clearly demonstrate elevated INF-g in liver extract of both dog groups, but five times lower than IL-10 in this organ; this suggests that IL-10 production in the liver is inhibiting INF-g making it the main regulatory cytokine in CVL. This elevated IL-10 production in asymptomatic and symptomatic dogs is similar to that seen by Ghalib et al. (1993), suggesting that the immune response in dogs is similar to that in human patients with acute VL. The high IL-10 production seen indicates that macrophages and T cells from infected dogs are producing IL-10 in response to the leishmania antigen and that this production could be important in the persistence of the parasite in host cells. Observation of raw IL-10 production data analysis shows a tendency towards higher production in symptomatic dogs, confirming studies in humans where disease severity and tissue lesion are associated with high serum levels of IL-10 (Gasim et al., 1998; Ghalib et al., 1993). Similarities observed in immunological response between naturally infected dogs and patients with acute VL suggest that the dog is an excellent model to study new therapies. In dogs inoculated with L. (L.) infantum amastigotes, Santos-Gomes et al. (2002) observed low proportion of animals which express IL-10 and suggested that this may not have a direct inhibitor effect on INF-g. Our
results were different; all studied dogs produced IL-10, and it is important to highlight the difference between natural and experimental infection, and that possibly, the initial immunomodulatory role of saliva on bleeding could play a fundamental in subsequent adaptive response (Paranhos et al., 1993). Our results indicate the coexistence of IL-10 and IFN-g in both dog groups naturally infected by L. (L.) chagasi similar to that seen in human VL (Ansari et al., 2006). The elevated IL-10 production could cause an alteration in macrophage activity, which could increase parasitemia and disease progress. INF-g, the cytokine related to VL resolution in humans and murine models (Wilson et al., 1998), produced less than the other cytokines studied. Our spleen extract results showed that symptomatic dogs produce higher quantities than the asymptomatic group. Disease chronicity in the dog seems to be associated to increased spleen IFN-g. The observation that high IFNg levels seem to be associated with worse disease prognosis in the dog indicates a failure in response to this cytokine, which is similar to a study on humans where the disease was prolonged by a lack of response to treatment (Ansari et al., 2006). Interestingly, that reduced expression of IFN-g R1 receptor in humans with VL has already been reported (Dasgupta et al., 2003) but there has been no study in dogs. Quinnell et al. (2001) using RT-PCR, considered that independently analysed levels of INF-g may not be good indicators of disease resistance, because similar levels of INF-g were found in tissue of both asymptomatic and symptomatic dogs. Our results were similar with this study, because INF-g production was seen in both groups of dogs. In dogs, isolated analysis of INF-g is not a good indicator of CVL status or eventual cure; this is similar to that suggested by Karp et al. (1993). Few studies have shown the profile of cytokines in dogs infected by L. (L.) chagasi; in this quantitative study we saw that asymptomatic dogs generally produced lower mean quantities of cytokines than symptomatic dogs; seen by the higher mean production levels of TGF-b1 and IL-10, supporting the observation of a predominance of type Th2 immune response, similar to that suggested by Santos-Gomes et al. (2002). Santos-Gomes et al. (2002) observed that Th1 response was capable of slowing down the start of the disease, but incapable of controlling it, and Chamizo et al. (2005) also observed that although both cytokine profiles are produced in asymptomatic dogs infected by Leishmania sp., only Th1 response gives immunity against the parasite. The results of this study
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