During Leishmania infection, tissue parasitism at different sites may differ and imply distinct immunopathological patterns during canine visceral leishmaniasis (CVL). For this reason, we have assessed by flow cytometry the impact of spleen and skin parasite density on the phenotypic profile of splenocytes and circulating leukocytes of 40 Brazilian dogs naturally infected by Leishmania chagasi categorized according to splenic and cutaneous parasite load. Our major statistically significant findings demonstrated that dogs with splenic high parasitism presented a significant decrease in absolute counts of CD5+ T lymphocytes in comparison with dogs presenting splenic medium parasitism. Moreover, a decrease in the absolute number of circulating monocytes was observed as a hallmark of high parasitism. The increased frequency of CD8+ T cells is associated with low splenic parasitism during CVL. Although we did not found any significant differences between the immunophenotypic analysis performed in circulating lymphocytes according to cutaneous parasite load, there were negative correlations between CD5+ and CD8+ T cells and cutaneous parasite density reemphasizes the role of T cell-mediated immune response in resistance mechanisms during ongoing CVL. These results add new insights about the pathogenesis of CVL and may help in the establishment of additional tools for future studies on drugs and vaccine approaches.
Visceral leishmaniasis (VL), which is caused by Leishmania (Leishmania) infantum [syn. Leishmania (Leishmania) chagasi], is endemic in over 88 countries within Europe and Latin America and is transmitted by the bite of the female sand fly (Desjeux 2004). The major prophylactic practice to control this human disease, as recommended by the World Health Organization, involves a systematic treatment of human cases besides vector control by insecticide and elimination of the domestic reservoir, mainly seropositive infected dogs (Tesh 1995). In this context, canine visceral leishmaniasis (CVL) has high epidemiological relevance, considering its increased incidence in the last decade, in addition to the intense cutaneous parasite density reported in both asymptomatic and symptomatic dogs which may contribute to the spread of disease (Molina et al. 1994; Tesh 1995; Giunchetti et al. 2006; Reis et al. 2006a, b).
Considering the pathological similarities between dogs and humans in many diseases caused by infectious agents, several studies have utilized these animals as an alternative model to evaluate parasite–host interaction, immune responses, and development of new methods focusing on the diagnosis, prognosis, and the evaluation of therapeutic and vaccine protocols for use in routine clinical veterinary (Cobbold and Metcalfe 1994; Williams 1997). Moreover, dogs constitute an excellent model to study leishmaniasis (Moreno and Alvar 2002; Alvar et al. 2004), and for this reason, the natural history of CVL has been recently described, particularly in relation to the parasite load in different tissues and to the immunopathological changes concerning the progression of clinical forms (Chamizo et al. 2005; Giunchetti et al. 2006; Reis et al. 2006a, b, c; Lage et al. 2007; Giunchetti et al. 2008a, b).
In this context, the clinical status of CVL is highly variable. Some infected dogs may develop symptomatic infection resulting in death, while others remain asymptomatic or develop one or more mild symptoms and are classified as oligosymptomatic (Mancianti et al. 1988; Barbiéri 2006; Reis et al. 2006a, b, c). Dogs infected with the Leishmania parasite present high titers of anti-Leishmania antibodies and a depression of the T cell-mediated response (Pinelli et al. 1994; Martinez-Moreno et al. 1995; De Luna et al. 1999; Campino and Abranches 2002).
Aiming to further characterize the immunopathological pattern in several animal species, flow cytometry has been shown to be a promising tool to evaluate a variety of cell subsets with a great interest and emphasis on studies in canine clinical veterinary (Reis et al. 2005). Moore et al. (1992) studied specific monoclonal antibodies for canine CD4 and CD8 that are expressed in T lymphocytes subpopulations and verified a high CD4 expression in canine neutrophils. Pinelli et al. (1995), in an immunophenotypic study of canine cells, observed a decrease of CD4+ and CD8+ lymphocytes in peripheral blood cells of dogs with VL. Moreover, Reis et al. (2006c) observed a lower frequency of circulating B cells (CD21+) and monocytes (CD14+) as important markers of severe CVL, whereas increased levels of B (CD21+) and T (CD4+ and CD8+) lymphocytes appeared to be the major phenotypic feature of asymptomatic disease.
Despite these findings regarding major lymphocytes subsets in dogs with different clinical forms of VL, there is a great gap in the understanding of the role of the tissue parasite density during CVL immune response. Recently, in a pioneer study, our group demonstrated that the spleen and skin are the most relevant sites of high parasitism during ongoing CVL (Reis et al. 2006b). The skin is the first point of contact with organisms of the genus Leishmania from sand fly vectors (Giunchetti et al. 2006) and its parasite density has been associated with a granulomatous inflammatory pattern in dogs with VL (Dos Santos et al. 2004). This tissue was considered by Abranches et al. (1991) to be an important reservoir compartment for parasites in healthy and sick Leishmania-infected dogs and the important role of dogs in VL transmission is supported by the high parasite loads found in the skin of infected animals (Deane and Deane 1962). The spleen is the main site of lymphoid cells interposed into the blood stream. Moreover, there is a large amount of circulating Leishmania antigens in the spleen, besides high local parasite density frequently in contact with splenocytes that lead to strong cellular and humoral immune responses (Reis et al. 2006b).
In this current study, we have assessed the impact of spleen/skin parasite density on the phenotypic profile of mononuclear cells in Brazilian dogs naturally infected by L. chagasi categorized according to three different parasite densities. Our major findings describe spleen parasite density as being related more closely to major phenotypic changes in peripheral blood leukocytes than skin during CVL. Moreover, the association between spleen parasitism and CD8+ T cells reemphasizes the role of T cell-mediated immune response in resistance mechanisms during ongoing CVL.
Materials and methods
Sixty mixed-breed adult dogs of both genders, 2–6 years old, were provided by the Control Zoonosis Center in Belo Horizonte City Council, Minas Gerais state, Brazil after clinical preselection and maintained in the kennels of the Institute of Biological Sciences of Federal University of Minas Gerais, Minas Gerais state, Brazil. Prior to the inclusion in this study, all animals were treated for intestinal helminthic (Endal plus®) infections and immunized against parvovirosis, leptospirosis, distemper, parainfluenza, and hepatitis (HTLP 5CV-L vaccine Pfizer®). The animals were kept in quarantine with drinking water and a balanced feed given ad libitum. The dogs used in this study were stray or domiciled mongrel dogs naturally infected with L. chagasi, selected based on their serological results on indirect immunofluorescence assay test (IFAT), used as a gold standard immunological test for diagnosis of CVL. Animals presenting IFAT titers ≥1:40 were considered positive and included into the L. chagasi-infected groups. Animals with IFAT negative (<1:40) were considered noninfected and included as a control group. Infection with L. chagasi was confirmed serologically in all IFAT-positive dogs, including enzyme-linked immunosorbent assay (ELISA) extract and ELISA r-K39, as described previously (Reis et al. 2005) and/or at least one parasitological examination performed at two different tissue sites (spleen and skin), as described below.
All procedures in this study were according to the guidelines set by the Brazilian Animal Experimental College (COBEA). This study was approved by the Ethical Committee for the use of Experimental Animals of the Federal University of Minas Gerais, Minas Gerais state, Brazil (CETEA).
Assessment of parasitological parameters was performed by diagnoses in tissue smears (spleen and skin) carried out after euthanasia performed by intravenous overdose of barbiturate and necropsy of the animals. The smears were stained by Giemsa and examined under optical microscopy for the identification of amastigote forms of Leishmania. Parasite density evaluation was performed in the spleen and skin and the results were expressed as Leishman Donovan units (LDU index), according to Reis et al. (2006b), modified from Stauber (1955) which corresponds to the number of Leishmania amastigotes by 1,000 nucleated cells. Tissue parasitisms for both sites were classified initially as low (LP), medium (MP), and high (HP) parasitism based on spleen- and skin-specific LDU values categorized statistically into tertiles according to Reis et al. (2006b), as follows: LP (zero to 11 in the spleen; zero to nine in the skin); MP (12–170 in the spleen; ten to 130 in the skin), and HP (184–2,564 in the spleen; 133–7,246 in the skin). The number of animals included on each subgroup was approximately 13–14 animals.
Noninfected dogs (CG, n = 20) displaying negative parasitological and serological examination for Leishmania were considered in this study as the control group.
Blood sample collection
Five milliliters of peripheral blood from the brachiocephalic vein were collected into tubes containing ethylenediamine tetraacetic acid (EDTA; final concentration of 1 mg/mL). A hemogram was performed in each sample (Coulter MD18; Luton, UK). All samples were maintained at room temperature up to 12 h prior to processing.
Immunophenotyping by flow cytometry
Immunophenotyping analyses of peripheral blood through flow cytometry were undertaken as described by Reis et al. (2005). Briefly, 1 mL of EDTA whole blood was submitted to prefixation and erythrocyte lysis by the slow addition of 13 mL of lysis solution (FACS lysing solution; Becton Dickinson, San Diego, CA, USA) followed by incubation for 10 min at room temperature (RT). After centrifugation (450×g for 10 min at RT), the pellet was resuspended in 500 μL phosphate-buffered saline (PBS) supplemented with 10% of fetal bovine serum (FBS-10%). In 96-well U-bottom plates (Limbro Biomedicals, Aurora, OH, USA), 30 μL of prefixed leukocyte suspension were incubated at RT for 30 min in the dark in the presence of 30 μL of anticanine cell surface marker monoclonal antibodies (mAbs) diluted previously in PBS-10% in indirect immunofluorescence procedures. A range of cell surface markers that define major canine leukocyte subpopulations were used, including diluted purified antidog CD5 1:800 (rat IgG2a, clone YKIX322 3), antidog CD4 1:12,500 (rat IgG2a, clone YKIX302 9), antidog CD8 1:800 (rat IgG1, clone YCATE55 9), anti-MHCII 1:200 (rat IgG2b, clone YKIX334 2), anti-CD45RA 1:200 (rat IgG2b, clone YKIX753 22 2), and anti-CD45RB 1:800 (rat IgG2b, clone YKIX716 13), all purchased from Serotec (Oxford, UK). Cells were additionally incubated in the same conditions, in the presence of 60 μL of previous diluted fluorescein isothiocyanate (FITC)-conjugated sheep antirat IgG antibody.
Five microliters of undiluted FITC-labeled mouse antihuman CD21 (mouse IgG1, clone IOBla; Immunotech, Marseille, France) and 50 μL of previously diluted PE/Cy-5-conjugated mouse antihuman CD14 1:200 (mouse IgG2a, clone TÜK4; Serotec Oxford, UK) were used in direct immunofluorescence procedures.
For the achievement of splenocytes, fragments of spleen were collected and transferred to a glass macerator. In this procedure, heparinized RPMI 1640 and Ficoll-Hypaque (Histopaque® 1.077-Sigma, USA) was used according to standardized protocol. In the immunofluorescence assay to evaluate the phenotypic expression of splenocytes obtained in the ex vivo context, 2–3 mL of the cellular suspension of each spleen were submitted to lysis, using the same solution described above.
Before flow cytometric data collection and analysis, labeled cells were fixed for 30 min with 200 μL of FACS FIX solution (10.0 g/L paraformaldehyde, 10.2 g/L sodium cacodylate, and 6.65 g/L sodium chloride, pH 7.2).
Flow cytometric acquisition and data analysis
Flow cytometric measurements were performed on a FACScan instrument (Becton Dickinson, Mountain View, CA, USA). The CellQuest software package was used in both data acquisition and analysis. A total of 10,000 events was acquired for each preparation. Canine whole blood leukocytes and splenocytes were identified on the basis of their specific forward (FSC) and side (SSC) light-scatter properties. Following FSC and SSC gain adjustments, the lymphocytes were selected based on their characteristic FSC versus SSC gain distribution. Fluorescence was evaluated based on the spectra of FITC and Cy5-PE on FL1 or FL3 single-histogram representation, respectively. The monocytes were analyzed by fluorescence intensity detection on single histograms directly on ungated leukocytes. For data analysis, a marker was set on the internal control in order to confine over 98% of the unlabeled cells.
The results were expressed in absolute counts (cell number per cubic millimeter) that allow the normalization of data from groups whose overall leukocytes counts may differ. The absolute counts for lymphocyte subsets were calculated as the product of the percentage of positive cells (CD5+, CD4+, CD8+, CD21+) within gated lymphocytes by the absolute lymphocyte counts derived from the hemogram. The absolute counts for monocytes were obtained as the product of CD14+ cells obtained within ungated leukocytes by the total white blood cell counts derived from the hemogram. Semiquantitative analyses were performed to evaluate differential expression of cell surface markers presenting unimodal distribution (MHC-II, CD45RA, and CD45RB). In these cases, the results were expressed as the mean fluorescence channel (MFC).
Statistical analysis was performed using the GraphPad Prism 4 0 3 software package (San Diego, CA, USA). In the parametric data, one-way analysis of variance was used for the comparative study between groups, followed by Tukey’s test. In the nonparametric data, Kruskal–Wallis test was used for between group comparative study, followed by Dunns’ test for multiple comparisons. Spearman’s rank correlation was also computed to investigate associations between expression of phenotypic features and spleen and skin parasite densities. In all cases, the differences were considered significant when the probabilities of equality, P values, were <0.05.
Results and discussion
Marked anemia, eosinopenia, and monocytopenia are hallmarks of hematological dysfunction associated with high spleen parasite density during canine visceral leishmaniasis
Recently, some studies focusing on the immunopathological pattern in CVL had been performed considering different tissues and distinct clinical forms (Chamizo et al. 2005; Giunchetti et al. 2006; Reis et al. 2006a, b, c; Lage et al. 2007; Giunchetti et al. 2008a, b). Although immunophenotypic changes in peripheral blood cells of dogs classified into different clinical forms have been previously reported, there are very few studies regarding these changes in dogs according to their parasite load in different organs (Sanchez et al. 2004; Reis et al. 2006c; Giunchetti et al. 2008b). It has been proposed that the spleen and the skin are the major sites of high parasite density during ongoing CVL (Reis et al. 2006b), and for this reason, the immunophenotypic alterations in dogs according to their splenic or cutaneous parasite load were analyzed in the present study.
The assessment of hematological parameters demonstrated severe anemia in dogs with splenic HP (Table 1). We observed significant decreases in erythrocytes of MP and HP dogs compared to the control group (p < 0.001) as well as in hemoglobin and hematocrit values in HP dogs compared to the LP group (p < 0.05) (Table 1). Dogs with high cutaneous parasite densities showed only decreased hemoglobin values in comparison to the LP dogs (p < 0.05). These data are in agreement with Reis et al. (2006c) which also observed these hematological alterations associated with severe disease. In addition, white blood cell counts of dogs with both parasitized compartments demonstrated significant decrease of eosinophils (p < 0.001) in MP and HP dogs as well as decrease in the absolute number of monocytes (p < 0.05) in HP dogs compared to the control group (Table 1). This finding is in accordance with a decrease (p < 0.05) in the absolute number of CD14+ cells observed in the HP group in comparison with the MP group (Fig. 1). According to Reis et al. (2006a, c), the decrease in absolute values of circulating monocytes in symptomatic and high parasitism dogs may suggest, during active CVL, the recruitment of these cells to lymphoid tissue where they might play an important role in immunological connections throughout antigen presentation. Giunchetti et al. (2006) showed a reduction in CD14+ monocytes counts in the skin of symptomatic dogs and a positive correlation between chronic dermal inflammation score and CD14+ monocytes, demonstrating a potential activation of this cell population and migration to the dermis, but with scant contribution to the resistance for L. chagasi infection.
Increased frequency of CD8+ T cells is associated with low splenic parasitism during CVL
In order to evaluate whether the parasite load during CVL reflects in the differential immunophenotypic profile of peripheral blood, we conducted an analysis of circulating leukocytes of L. chagasi-infected and control dogs. Our data demonstrated that dogs with splenic HP presented a significant decrease (p < 0.05) in absolute counts of CD5+ T lymphocytes in comparison with the MP group (Fig. 1). Moreover, analysis of the CD5+/CD21+ cell ratio revealed an increase (p < 0.05) in the LP and MP groups compared to the control group.
Analysis of T cell subpopulations showed a decrease (p < 0.05) in CD4+ T lymphocytes in the HP group when compared to the MP group and an increase (p < 0.05) in CD8+ T lymphocytes in the LP and MP groups compared to the control group (Fig. 1). In order to explore more effectively the balance between the T cell subpopulations, we have also reported a decreased (p < 0.05) CD4+/CD8+ cell ratio in infected dogs presenting LP and MP parasitism compared to the control group (Fig. 1), reinforcing the role of CD8+ T cells in tissue parasite control.
Typically, high CD8+ T cells counts have been observed in the peripheral blood of asymptomatic dogs and are associated with low bone marrow parasitism, suggesting that these subpopulations create a microenvironment efficient to remove the parasites in hosts bearing asymptomatic or mild disease and with lower parasite densities (Reis et al. 2006c). In addition, CD8+ T cells are thought to play an important role in the development of an effective immune response to Leishmania sp., possibly through cytotoxic mechanisms, which act as potent protective events during CVL (Pinelli 1997). In fact, different studies have correlated the level of CD8+ T cells with protection during L. infantum/L. chagasi infection (Pinelli 1997; Reis et al. 2006c) or during the immunogenic response after vaccine administration against CVL (Giunchetti et al. 2007; Giunchetti et al. 2008c).
Expression of MHC-II, CD45RA, and CD45RB by peripheral blood lymphocytes was evaluated through semiquantitative analyses in order to verify whether parasite load may be associated with an altered pattern of these constitutive cell surface markers. Our data did not show any statistically significant differences for MHC-II or CD45RA and CD45RB considered as isolated parameters (Fig. 1) or even in the CD45RB/CD45RA expression index (data not shown). Although important differences in the expression of these markers were already found by our group when we evaluate them according to distinct clinical forms of CVL (Reis et al. (2006c), herein, we did not find any statistically significant differences for these cell markers, suggesting that these parameters were not influenced by splenic or cutaneous parasite density during CVL.
High splenic parasite density is correlated with a lower frequency of circulating T cell subsets
To confirm and extend our findings, we performed correlation analyses between the expressions of major lymphocyte subsets counts in the peripheral blood with splenic parasite density (Fig. 2). Our data showed an association between low cell counts and high spleen parasite densities during CVL. Negative correlations between T cell subsets (CD5+, r = −0.4344, p = 0.0057; CD4+, r = −0.3223, p = 0.0454; and CD8+ cells, r = −0.5012, p = 0.0012) as well as the CD5+/CD21+ cell ratio (r = −0.3457, p = 0.0311) and spleen parasite density were observed. We found either a positive correlation between CD4+/CD8+ cell ratio (r = 0.5455, p = 0.0003) and spleen parasite density (Fig. 2).
Augmented skin parasite density is related to the low levels of circulating CD8+ T cells besides high CD4+/CD8+ cell ratio
The immunophenotyping of peripheral blood lymphocytes from dogs categorized according to their skin parasite load did not show any statistically significant differences between the groups (Fig. 3). However, the correlation analyses between expression of phenotypic features and skin parasite densities showed negative correlations for CD5+ T cells (r = −0.3218, p = 0.0429) and CD8+ T cells (r = −0.3411, p = 0.0312), indicating that, similar to the results obtained with splenic parasitism, dogs with highest cutaneous parasite densities have the lowest levels of both these phenotypes. A positive correlation between the CD4+/CD8+ cell ratio (r = 0.3709, p = 0.0185) and skin parasite density was also found (Fig. 4), according to the previous finding that reported a positive correlation between chronic dermal inflammation score in CVL and circulating CD4+ T cells (Giunchetti et al. 2006).
In order to evaluate the association between skin and spleen parasite load, we have performed a correlation between splenic and cutaneous parasite density, demonstrating the presence of a significant positive correlation (r = 0.818, p = 0.000) between these two tissues parasite densities during CVL (data not shown).
Increased proportions of CD8+ and decreased proportions of CD45RA+ splenocytes are associated with medium splenic parasitism during CVL
During Leishmania infection, there is a large amount of circulating parasite-derived antigens that reach the spleen. In addition, the high local parasite density frequently in contact with splenocytes would lead to a cellular activation and a strong immune response to the parasites (Reis et al. 2006b). In order to analyze the compartmentalized immune response in the spleen, we have performed an immunophenotypic characterization of major splenic lymphocyte populations from L. chagasi-infected or control group according to their spleen parasite density (Fig. 5). Our data revealed a significant increase (p < 0.05) in CD8+ splenocytes in the MP group compared to the control group (Fig. 5). We postulate that CD8+ splenocytes may present a distinct activation status during CVL, possibly associated with immunomodulatory or suppressor cell activity. According to this hypothesis, Peruhype-Magalhães et al. (2006) showed, in human active visceral leishmaniasis, that circulating CD8+ T cells showed a mixed cytokine pattern characterized by elevated levels of both intracellular IFN-γ and IL-10. Similar results displaying a mixed pattern of cytokine mRNA in splenocytes during CVL has been published by our group (Lage et al. 2007). In this study, the levels of INF-γ and IL-10 expressions were positively correlated with the parasite load. In addition, Strauss-Ayali et al. (2007) also observed that both type 1 and type 2 immune responses occur in the spleen during canine L. infantum infection.
Furthermore, we observed a significant decrease (p < 0.05) in the CD45RA expression in the MP group compared to the control group (Fig. 5). According to Tipold et al. (1998), loss of CD45RA was shown to occur in other species upon T cell activation. Therefore, the decrease in the CD45RA expression might suggest that, in the spleen of dogs with medium parasite density, there is still an activation profile of T cells. However, whether the changes in CD45 isoforms expression occur simultaneously or sequentially in different tissues, and how they may affect the development of CVL, needs to be determined.
In conclusion, our findings highlight the importance of quantitative investigations regarding the parasite load at different tissues associated with immunopathological changes during ongoing CVL. The assessment of dogs categorized according to parasite density may contribute as an additional tool for immunological investigations, considering therapeutic and vaccine approaches.
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The study was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil (FAPEMIG grant: CBB CBB9202), PAPES III-B (FIOCRUZ/RJ/2002), and CNPq. OAMF, RCO, and ABR are grateful to CNPq for fellowships. The authors wish to express their particular appreciation for the hard work carried out by the kennel staff of the Federal University of Ouro Preto and for their dedication during the execution of this project. All procedures in this study complied with the current laws of Brazil according to the guidelines set by the Brazilian Animal Experimental College (COBEA).
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Guerra, L.L., Teixeira-Carvalho, A., Giunchetti, R.C. et al. Evaluation of the influence of tissue parasite density on hematological and phenotypic cellular parameters of circulating leukocytes and splenocytes during ongoing canine visceral leishmaniasis. Parasitol Res 104, 611 (2009). https://doi.org/10.1007/s00436-008-1237-4
- Visceral Leishmaniasis
- Parasite Density
- Parasite Load
- Absolute Count