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c mice infected by Leishmania infantum
Experimental Parasitology 207 (2019) 107789
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CXCL10 treatment promotes reduction of IL-10+ regulatory T (Foxp3+ and Tr1) cells in the spleen of BALB/c mice infected by Leishmania infantum
T
Webertty Mayk Eufrásio de Figueiredoa, Fabiola Fernandes Herediaa, Aline Sombra Santosa, Roberta da Rocha Bragab, Francisco Rafael Marciano Fonsecac, Naya Lúcia de Castro Rodriguesc, Ticiana Monteiro Abreua, Margarida Maria de Lima Pompeua, Helene Santos Barbosad, Maria Jania Teixeiraa,∗ a
Federal University of Ceará, Department of Pathology and Legal Medicine, 60441-750, Fortaleza, CE, Brazil Federal University of Ceará, Department of Biology, 60440-900, Fortaleza, CE, Brazil Federal University of Ceará, Faculty of Pharmacy, Dentistry and Nursing, Department of Pharmacy, 60430-170, Fortaleza, CE, Brazil d Oswaldo Cruz Foundation, Oswaldo Cruz Institute, Laboratory of Structural Biology, 21040-360, Rio de Janeiro, RJ, Brazil b c
A R T I C LE I N FO
A B S T R A C T
Keywords: CXCL10 Leishmania infantum Treg cells
American visceral leishmaniasis is caused by the protozoan Leishmania infantum. The control of the disease depends on the magnitude of the Th1 cell response and IL-10 producing regulatory T cells. Administration of chemokine, such as CXCL10, has shown promising results in the leishmaniasis treatment. Previous studies from our group have shown that CXCL10 induces a reduction in parasite burden in the spleen and a decrease in IL-10 and TGF-β production in L. infantum-infected BALB/c mice. This work investigated whether CXCL10-treatment reduces IL-10 + Treg cell populations (CD4+CD25+Foxp3+ and Tr1) and induces morphological changes in the spleen. BALB/c mice were infected and treated or not with CXCL10 on the 1st, 3rd and 7th days of infection. CXCL10-treatment was able to reduce the parasite load in the spleen in L. infantum-infected BALB/c mice and this decrease in the number of parasites correlated with the decrease in size of this organ in treated animals compared to untreated animals. 7, 23, and 45 days post-treatment (p.t.), the phenotype and frequency of IL10 + Treg cells were evaluated by flow cytometry, and the morphological changes of the spleen were analyzed by optical microscopy. After 7 and 23 days p.t., CXCL10-treated animals showed a significant reduction of CD25Foxp3−IL-10+ (Tr1) cells in the spleen when compared to untreated animals, whereas CD4+CD25+Foxp3+IL10+ Treg cells reduced later at 23rd and 45th days p.t. Furthermore, while untreated animals showed a significant positive correlation between IL-10 production and Tr1 cells, in CXCL10-treated group this correlation was negative. Thus, these findings show that treatment with CXCL10 chemokine in L. infantum-infected BALB/c mice results in suppression of IL10+ Treg (Foxp3+ and Tr1) cells in the spleen, associated with a reduction in parasite load and splenomegaly.
1. Introduction
et al., 2006; Santos et al., 2008). In mice models of VL, the control of the disease depends on the magnitude of the Th1 cell response, which leads to the production of IFN-γ whose pro-inflammatory action is fundamental (Kaye et al., 2004). IL-10 is an immunoregulatory cytokine present in high levels during the disease active stage, and the association of immunosuppression induced by IL-10 in human VL is well established
Visceral leishmaniasis (VL) affects 500,000 people directly and about 350 million people are at risk, including Brazil (Pace, 2014; WHO, 2017) whose disease is caused by L. infantum (Maurício et al., 2000). Treatment of this disease causes, among other problems, toxic effects and resistance of strains to the drugs currently in use (Croft
∗ Corresponding author. Department of Pathology and Legal Medicine, Federal University of Ceará, Alexandre Baraúna Street, 949, Fortaleza, CE, 60430-160, Brazil. E-mail addresses: [email protected] (W.M. Eufrásio de Figueiredo), [email protected] (F.F. Heredia), [email protected] (A.S. Santos), [email protected] (R. da Rocha Braga), [email protected] (F.R. Marciano Fonseca), [email protected] (N. Lúcia de Castro Rodrigues), [email protected] (T.M. Abreu), [email protected] (M. Maria de Lima Pompeu), [email protected] (H.S. Barbosa), [email protected] (M.J. Teixeira).
https://doi.org/10.1016/j.exppara.2019.107789 Received 13 June 2019; Received in revised form 22 October 2019; Accepted 22 October 2019 Available online 24 October 2019 0014-4894/ © 2019 Elsevier Inc. All rights reserved.
Experimental Parasitology 207 (2019) 107789
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recovered from the animal, the parasites were cultured at 25 °C in N.N.N. medium, containing Schneider's insect medium (Sigma-Aldrich) supplemented with 20% fetal bovine serum (Sigma-Aldrich), 2% sterile human urine and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) (all Sigma-Aldrich). For infection, the parasites were used until the 5th passage in vitro.
(Gupta et al., 2011; Owens et al., 2012; Resende et al., 2013). Regulatory T (Treg) lineage (nTreg and iTreg) is a cell subset regulatory that exhibits forkhead boxP3 (Foxp3) as its transcription factor and regulates Th1 response. The peripheral population of Foxp3+ Treg cells comprises both nTreg and iTreg cells (Curotto de Lafaille and Lafaille, 2009). In addition to the Foxp3+ Treg cells, there are other types of CD4+ Treg cells named Foxp3- type 1 regulatory T (Tr1) cells. These cells are induced in the periphery and have a pivotal role in promoting and maintaining T cell tolerance in BALB/c mice (Yao et al., 2015). In previous studies in Leishmania-infected BALB/c mice, Treg cells presented different roles according to the species of Leishmania used in the infection, contributing either to the pathology and persistence or to resolution of the disease. In the case of L. donovani, evidence indicates that the induction of Treg cells results in an aggravated infection (Martin et al., 2010). In the spleen, the main characteristic of chronic VL is the breakdown of the splenic architecture. In the presence of Leishmania, TNF-α starts to be produced in large quantities triggering production of IL-10 and depletion of the marginal zone (MZ) macrophages (Engwerda et al., 2002; Santana et al., 2008). The deletion of follicular dendritic cells (FDC) and the germinal centers (GC) disorganization prevent the longterm interactions between B cells and T follicular cells necessary for the generation of specific antibodies, that frustrate the action of the parasite (Victora and Nussenzweig, 2012). Exogenous CXCL10 treatment in L. major-infected BALB/c mice has been shown to induce strong recruitment and activation of NK cells (Vester et al., 1999; Muller et al., 2001). Also, treatment with CXCL10 in L. amazonensis-infected BALB/c mice may decrease lesion size and parasitic burden, accompanied by increased production of INF-γ, IL-12 and NO (Vasquez and Soong, 2006). Studies with L. donovani-infected BALB/c mice have demonstrated that CXCL10 is involved in the protective response of infection, inducing a Th1 response, by regulating the pathway of inflammatory mediators such as NO and pro-inflammatory cytokines (Gupta et al., 2009). CXCL10 may also participate in protection against L. donovani with a marked decrease of immunoregulatory cytokines secreted by Treg cells (Gupta et al., 2011). Previous studies of our group have shown an important protective role of CXCL10 in L. infantum-infected BALB/c mice mediated by a significant production of IFN-γ and suppression of the immunoregulatory cytokines IL-10 and TGF-β, leading to the hypothesis if it is not associated with a decrease in the frequency of regulatory T cells (Figueiredo et al., 2017). Therefore, in the present study, we assessed whether treatment with CXCL10 would confer protection against L. infantum infection by regulating the induction of IL10+ Treg cell populations (CD4+CD25+Foxp3+ and Tr1) and also whether treatment with CXCL10 would have repercussions on the changes that occur in the spleen, the organ considered responsible for the persistence of the infection in the visceral leishmaniasis.
2.3. Infection and treatment with CXCL10 Metacyclic promastigotes at the stationary phase were inoculated intraperitoneally (ip.) into BALB/c mice at a concentration of 2 × 107 parasites in 100 μL of sterile saline (NaCl 0.9%). The animals were divided into two groups: one group received CXCL10 (5 μg/kg) (Thermo Scientific) and the other received saline (untreated group) ip. after 1, 3 and 7 days of infection (Gupta et al., 2009). For the histological analysis, an additional uninfected group was used as negative control. The animals were euthanized after 7, 23 and 45 days of treatment by halothane's inhalation (Sigma-Aldrich). For parasitic load analysis, a group of animals was also euthanized on the first day p.t. Six animals per group were euthanized at each time period. The animals were weighed before starting treatment and before euthanasia for weight analysis.
2.4. Parasite load The number of parasites was quantified in the spleen (Titus et al., 1985). The animals were euthanized by inhalation of halothane (SigmaAldrich) and submerged in 3% iodized alcohol up to 3 min to allow decontamination. Then the spleen was removed aseptically, weighed, and one fragment of the spleen (from 0.0671 to 0.0700 g) was homogenized in 1 mL of Schneider medium and left to rest for 5 min. Eight dilutions were made from this cell suspension (10, 50, 100, 500, 103, 104, 105 and 106) in Schneider supplemented with 20% FBS (SigmaAldrich), 2% sterile human urine and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). One hundred microliters of these dilutions were distributed into 96-well plates, flat bottom, 6 wells/dilution. The plates were sealed and incubated at 25 °C for 3 weeks. The wells were observed on an inverted optical microscope (Nikkon) every 3 days to record the dilutions containing promastigotes. The final number of parasites was determined using the ELIDA software (Taswell, 1984).
2.5. Flow cytometry Single cell suspensions were obtained from spleens of animals infected with L. infantum promastigotes, treated or untreated with CXCL10 after seven, 23 and 45 treatment days. For this, each animal spleen was macerated on a nylon filter with pores (70 μm; Falcon) coupled to a 50 mL tube. The obtained cell filtrate was suspended in RPMI medium and subjected to centrifugation (300×g; 5 min; 4 °C). Posteriorly, for regulatory T cell phenotyping, CD4+CD25+Foxp3+IL10+ Treg cells were stained with PerCP Rat Anti-Mouse CD4 (0.2 μg/ test; clone RM4-5), FITC Rat Anti-mouse CD25 (0.5 μg/test; clone 7DA), PE Anti-Mouse/Rat Foxp3 (0.2 μg/test; clone FJK-16s) and APC Rat Anti-Mouse IL-10 (REF: 554468) (all BD Biosciences). For intracellular staining, the cells were permeabilized with a FIX/PERM Kit (eBioscience), according to the manufacturer's guide. For all analyses, the results were compared to those obtained with cells stained with the isotype control antibodies rat IgG2b (A95-1) and rat IgG2a (R35-95) (BD Biosciences, San Diego, CA, USA). After staining, cells were fixed in 1% paraformaldehyde. Cell acquisition was performed using a FACS Calibur flow cytometer and Cell Quest software (BD Biosciences) on at least 30,000 events. Data were plotted and analyzed using Cell Quest (BD Biosciences) and WinMDI 2.9 software (Joseph Trotter, la Jolla, CA, USA).
2. Methods 2.1. Animals Male BALB/c mice, 6–8 weeks old, weighing 18–20g were obtained from the Pathology and Legal Medicine Department of the Federal University of Ceará (DPML/UFC). The animals were housed under specific pathogen-free conditions (12 h light/12 h dark cycle; 22–24 °C), with appropriate commercial feed and water ad libitum. This study was conducted with the Committee on Ethics in the Use of Animals of the UFC approval (CEUA n° 52/2014). 2.2. Parasites The virulence of the L. infantum (MHOM/BR/BA-262) strain was maintained by regular passage in a male golden hamster. After being 2
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parasites in the treated group and this reduction remained after 45 days of treatment (Fig. 1B).
2.6. Histopathological analysis The animals were euthanized by halothane's inhalation in a hermetic chamber, after 7, 23 and 45 treatment days (n = 6), at each time period and from each group). Immediately after euthanasia, the mice were submitted to laparotomy for collecting the spleen. Each spleen was separately weighed to calculate the relative weight (spleen weight/ body weight). The spleens were fixed in 10% buffered formaldehyde, processed and, then, 5 μm microsections of the samples were made and stained with hematoxylin-eosin (H & E). Photomicrographs of 10 fields of the spleen were taken in a 400x magnification to analyze the white pulp. White pulp area and total spleen fragment area were measured digitally by ImageJ® v.1.44 software to analyze the percentage of white pulp.
3.2. Frequency of CD4+CD25+Foxp3+IL-10+ Treg cells in the spleen The gating strategy of Treg (CD4+CD25+Foxp3+) proportion and their expression levels of the cytokine IL-10 can be seen in Fig. 2A, B and 2C. After 7 days of treatment, there was no difference in the frequency of these cells between the CXCL10-treated and untreated animals. However, in the last two post-treatment periods (23 and 45 days) the animals treated with CXCL10 induced a lower frequency of CD4+CD25+Foxp3+IL-10+ Treg cells, compared to untreated animals (day 23, p = 0.0159; day 45, p = 0.0079) (Fig. 2D). In the CXCL10treated animals, the frequency of these cells reached its lowest value (approximately 15%) at day 45 post-treatment, which was equivalent to a reduction of approximately half, when compared to the frequency at day 23 post-treatment that had been 30% (p = 0.0204) (Fig. 2D). On the contrary, in the untreated group, the cell frequency increased between days seven and 23 post-treatment (p = 0.0013), and then it remained steady without any significant decrease between days 23 and 45 post-treatment (Fig. 2D). In addition, a significant correlation between IL-10 production and CD4 +CD25+Foxp3+IL-10+ Treg cells was not observed in the analyzed groups (Fig. 3A and B).
2.7. Statistical analysis The t-Student test was applied to verify the statistical significance between the treated and untreated groups. Comparisons in more than two groups were performed using one-way analysis of variance (ANOVA), followed by Bonferroni post-test. The tests were performed using GraphPad Prism Software version 6.00 (GraphPad Software, San Diego, CA, USA). The results are presented as the arithmetic mean ± mean standard error or as median (whiskers min to max). The minimum significance for all tests was accepted when p < 0.05.
3.3. Frequency of CD4+CD25-Foxp3−IL-10+ Treg (Tr1) cells in the spleen 3. Results
The gating strategy of Treg Tr1 (CD4+CD25-Foxp3-) proportion and their expression levels of the cytokine IL-10 can be seen in Fig. 4A, B and 4C. After 7 treatment days, the frequency of CD4+CD25-Foxp3−IL10+ Tr1 cells in the CXCL10-treated animals was lower when compared to untreated animals (p = 0.0195), and after 23 days of treatment, this frequency remained lower in the CXCL10-treated group (Fig. 4D). After 45 days of treatment, there was no difference in the frequency of these cells between the analyzed groups (Fig. 4D). Farther, CXCL10 treated group showed a significant negative correlation between IL-10 production and Treg Tr1 cells (r2 = 0.518, p = 0.008), while a significant positive correlation was observed in untreated animals (r2 = 0.5109, p = 0.0134) (Fig. 2C and D).
3.1. Relative weight and parasite load in the spleen In the analysis of the relative weight of the spleen was observed that on the 1st day after treatment, the treated group and the control group showed no difference in the weight of the spleen, although the weight of the spleen of both groups (control: 0.0056 ± 0.003; treated: 0.0045 ± 0.0004) was slightly higher than those found in healthy animals (0.0042 ± 0.0002) (Fig. 1A). When the treated group was analyzed separately, there was a decrease in spleen weight of the animals after the 7th day, unlike what happened with the control group, whose weights only declined after 45 days of treatment, and yet were above the weight of the spleen of healthy animals. When the groups were compared, it was observed that there were differences between them on day 7 (p = 0.0286) and 23 (p = 0.0033) after treatment (Fig. 1A). Regarding the number of parasites in the spleen, it was observed that after the 1st day of treatment, animals treated with CXCL10 showed no difference in the number of parasites when compared to control animals (Fig. 1B). After 7 days of treatment, the number of parasites in treated animals remained stable (approximately 104). After 23 days of treatment, there was a decline significant in the number of
3.4. Repercussion of CXCL10 treatment in the spleen CXCL10-treated animals showed a reduced splenomegaly when compared with untreated animals, both at 7 (p = 0.0286) and at 23 days post-treatment (p = 0.0033) (Fig. 5B). However, in relation to the area of white pulp there was no significant difference between the groups non-infected, CXCL10-treated and untreated (Fig. 5A and C and Table 1). Fig. 1. Relative weight (A) and Parasite load (B) in the spleen from BALB/c mice infected by L. infantum and treated with CXCL10. The animals were treated with CXCL10, intraperitoneally (5 μg/kg), or saline at 1, 3 and 7 days of infection. 1, 7, 23 and 45 days after the end of treatment the animals were euthanized and the spleen was removed and used for evaluating the relative weight and parasite load. Data from 4 to 6 animals per group are represented by arithmetic mean ± standard error. Significant differences among groups within the same time period were determined by t-Student test (p < 0.05); differences among the same groups in different time periods were determined by ANOVA (p < 0.05). The dashed lines represent the arithmetic mean of the relative weight of spleen (0.0042) of 6 healthy animals. 3
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Fig. 2. Gating strategy of proportion and frequency of regulatory T cells (CD4+CD25+Foxp3+) and their expression levels of cytokine IL-10 in splenocytes from BALB/c mice infected with L. infantum and treated or untreated with CXCL10. Lymphocytes were identified based on their forward - FSC - and side scatter characteristic - SSC (A). Within the lymphocyte population, CD4+CD25+ cells (B) were gated (R2) and the subpopulation of FoxP3+IL-10+ (C) quantified (R3). Regulatory T cells frequency from at least 6 animals per group are shown as the arithmetic mean ± standard error of the mean, and are representative of 3 independent experiments (D).
Fig. 3. Evaluation of the correlation between IL-10 production and Treg cells. A and B: correlation between IL-10 production and CD4+CD25+Foxp3+IL-10+ Treg cells frequency from untreated and CXCL10-treated BALB/c mice. C and D: correlation between IL-10 production and CD4+CD25-Foxp3−IL-10+ Tr1 cells frequency from untreated and CXCL10-treated BALB/c mice. 4
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Fig. 4. Gating strategy of proportion and frequency of Tr1 cells (CD4+CD25− Foxp3-) and their expression levels of cytokine IL-10 in splenocytes from BALB/c mice infected with L. infantum and treated or untreated with CXCL10. Lymphocytes were identified based on their forward - FSC - and side scatter characteristic - SSC (A). Within the lymphocyte population, CD4+CD25− cells (B) were gated (R2) and the subpopulation of Foxp3−IL-10+ (C) quantified (R3). Tr1 cells frequency from at least 6 animals per group are shown as the arithmetic mean ± standard error of the mean, and are representative of 3 independent experiments (D).
4. Discussion
suggesting a minor role of these cells during visceral L. infantum infection. On the other hand, the same study showed that Tr1 cells were an important source of IL-10, since they were in fact capable of specifically producing large amounts of this cytokine, early on and throughout the experimental period (Rodrigues et al., 2009). Herein, the frequency of Treg Tr1 cells in the CXCL10-treated animals was lower when compared to untreated animals at 7- and 23-days post-treatment. These data suggest that CXCL10 was able to act earlier to regulate this Tr1 regulatory population. Tr1 cells may be found two weeks after infection, producing both IFN-γ and IL-10, and reach a plateau in one month, representing 2–5% of the total splenic CD4+ T cell pool (Owens et al., 2012; Resende et al., 2013). The polarization for this population of CD4+T-bet+IFN-γ+IL-10+ T lymphocytes (Foxp3and CD25−) is associated with increased host susceptibility to Leishmania and initially depends on its activation only by infected dendritic cells known to have low levels of costimulatory molecules on their surfaces and a capacity to produce high amounts of IL-10 (Resende et al., 2013). The persistence of L. infantum in the spleen has a complex immunopathology and is still unknown in its particularities, since it involves several cells of the reticuloendothelial system, cytokines and chemokines. This persistence occurs due to the inability of the host to control the parasite load and to maintain the pro-inflammatory Th1 immune profile; this is due in part to the IL-10 production stimulated by the parasites (Moore et al., 2001). The spleen is a site of active infection and produces several hematological abnormalities in the host, causing the typical symptoms of infection, among them, splenomegaly directly related to the lymphoid follicle hyperplasia of the white pulp (Engwerda et al., 2002; Moore et al., 2001). Visceral leishmaniasis causes changes in the spleen normal physiology through a deep remodeling (Engwerda et al., 2002), with the loss of cellular organization in its intricate microarchitecture. The spleen has several populations of macrophages and structures (germinal centers) that allow antigenic presentation to activate effector cells, T and B lymphocytes (Allen et al., 2007). The MZ macrophages is compromised during infection by L. donovani, generating host immunosuppression (Engwerda et al., 2002; Santana et al., 2008). The next step of our study was to investigate how CXCL10-treatment could affect the spleen. Indeed, CXCL10-treated animals showed a
CXCL10, an IFN-γ-inducing chemokine, is a cytokine that in recent years has been explored in the field of immunotherapy as a potential drug for leishmaniasis. The CXCL10 effects, such as leukocyte activation, balance between regulatory and effector T cells, chemotaxis, hematopoiesis and increased natural killer (NK) cell cytotoxicity (Gupta et al., 2011) may all be of great benefit for the leishmaniasis treatment. Herein, we assessed whether treatment with CXCL10 would confer protection against L. infantum infection by regulating the induction of IL10+ Treg (CD4+CD25+Foxp3+ and Tr1) cell populations and also whether treatment with CXCL10 would have repercussions on the changes that occur in the spleen. In the present study, it was observed that CXCL10-treatment was able to reduce the parasite load in the spleen, four weeks after infection, representing a significant decrease in the number of parasites in this organ. This reduction in the number of parasites in the spleen correlated with the decrease in size of this organ in treated animals compared to control animals. Previous studies have shown that during infection by L. infantum in mice, the parasites multiply rapidly during the first 4 weeks in the liver and disappear spontaneously around the 8th week of infection, whereas in the spleen parasites grow more slowly, leading to a chronic infection in the organ (Wilson et al., 1996). CXCL10 treatment has been shown to induce, in the murine model, a higher production of Th1 cytokines, such as IFN-γ and IL-12, and a reduction of the suppressor cytokines IL-10 and TGF-β levels (Gupta et al., 2009, 2011; Figueiredo et al., 2017). IL-10 and TGF-β are produced by Th2 and Treg cells acting on immunosuppression, which aggravates visceral leishmaniasis (Gupta et al., 2011). In the present study, the CXCL10-treated animals presented the CD4+CD25+Foxp3+ Treg cells producing IL-10 frequency significantly lower in the last two post-treatment periods (23 and 45 days) when compared to the untreated group. These results corroborate with a study which showed that CXCL10 treatment results in protection in vivo against L. donovani, through a significant reduction of the immunosuppressive cytokines IL-10, due to modulation of regulatory T cells (Gupta et al., 2011). Unlike our results, some studies have shown that CD4+CD25+Foxp3+ T cells from L. infantum-infected BALB/c mice were able to secrete some IL-10 levels but nothing very expressive, 5
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Fig. 5. Repercussion of CXCL10 treatment in the spleen. White pulp area (A), spleen weight index (B), and spleen histological sections (C) of BALB/c mice infected with L. infantum treated or untreated with CXCL10; and non-infected. The animals were treated with CXCL10 (5 μg/kg) or sterile saline, ip., on the 1st, 3rd and 7th days of infection. After 7, 23 and 45 days of treatment, the animals were euthanized to collect the spleen. Data from at least 6 animals per group are shown as median (whiskers min to max) and are representative of 2 independent experiments. Histological sections: original magnification ×400.
inoculum is killed by MZ macrophages within the first 24 h after infection (Allen et al., 2007). CXCL10 induces IFN-γ secretion (Gupta et al., 2009, 2011; Figueiredo et al., 2017), produced by NK cells, which is an important cytokine that induces macrophage activation (Alexander, 2005; Dey et al., 2007). Other studies have pointed to the increased cytotoxicity of NK cells in BALB/c mice infected with L. major and treated with CXCL10 (Dey et al., 2007), which suggest that this cytotoxicity could help maintain the spleen structure by eliminating infected macrophages.
Table 1 White pulp area in relation to total spleen area (%) in BALB/c mice infected with L. infantum and treated with CXCL10. Time (days post-treatment)
7 23 45
Infected CXCL10
UNTREATED
30.3 24.9 37.0
36.5 27.7 38.4
P
ns ns ns
ns: no significant.
5. Conclusion reduced splenomegaly when compared with untreated animals. However, in relation to the area of white pulp there was no significant difference between the groups. About 50% of the initial parasite
In conclusion, the findings of this study, associated with a lower spleen weight and a reduction the parasite load in the spleen in the CXCL10-treated animals, suggests a protective role of CXCL10 6
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chemokine with suppression of IL10+ Treg (Foxp3+ and Tr1) cells in the spleen in L. infantum-infected BALB/c mice. These data may provide information for the development of future therapeutic interventions, using CXCL10 as an adjuvant in the visceral leishmaniasis.
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Funding This work was supported by Brazilian grants from Conselho Nacional de Pesquisa (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and CAPES-Programa DINTER (Doutorado Interinstitucional) Medicina Tropical 309/2013. Availability of data and materials All data generated or analyzed during this study are included in this published article. Declarations of competing interest None. Acknowledgements The authors wish to thank Dr. Andrea Henrique Pons of the Laboratory of Innovations in Therapies, Education and Bioproducts, Oswaldo Cruz Institute, and Dr. Adriano Gomes da Silva of the Evandro Chagas National Institute of Infectology, for their scientific advice on Treg cells and flow cytometry. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.exppara.2019.107789. References Alexander, J., 2005. Bryson K. T helper (h)1/Th2 and Leishmania: paradox rather than paradigm. Immunol. Lett. 99 (1), 17–23. Allen, C.D., Okada, T., Cyster, J.G., 2007. Germinal-center organization and cellular dynamics. Immunity 27 (2), 190–202. Croft, S.L., Sundar, S., Fairlamb, A.H., 2006. Drug resistance in leishmaniasis. Clin. Microbiol. Rev. 19 (1), 111–126. Curotto de Lafaille, M.A., Lafaille, J.J., 2009. Natural and adaptive foxp3+ regulatory T cells: more of the same or a division of labor? Immunity 30 (5), 626–635. Dey, R., Majumder, N., Bhattacharyya, M.S., Bhattacharjee, S., Banerjee, S., Roy, S., et al., 2007. Induction of host protective Th1 immune response by chemokines in Leishmania donovani-infected BALB/c mice. Scand. J. Immunol. 66 (6), 671–683. Engwerda, C.R., Ato, M., Cotterell, S.E., Mynott, T.L., Tschannerl, A., Gorak-Stolinska, P.M., et al., 2002. A role for tumor necrosis factor-alpha in remodeling the splenic marginal zone during Leishmania donovani infection. Am. J. Pathol. 161 (2), 429–437. Figueiredo, W.M.E., Viana, S.M., Alves, D.T., Guerra, P.V., Coêlho, Z.C.B., Barbosa, H.S.,
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CXCL10 treatment promotes reduction of IL-10+ regulatory T (Foxp3+ and Tr1) cells in the spleen of BALB/c mice infected by Leishmania infantum
T
Webertty Mayk Eufrásio de Figueiredoa, Fabiola Fernandes Herediaa, Aline Sombra Santosa, Roberta da Rocha Bragab, Francisco Rafael Marciano Fonsecac, Naya Lúcia de Castro Rodriguesc, Ticiana Monteiro Abreua, Margarida Maria de Lima Pompeua, Helene Santos Barbosad, Maria Jania Teixeiraa,∗ a
Federal University of Ceará, Department of Pathology and Legal Medicine, 60441-750, Fortaleza, CE, Brazil Federal University of Ceará, Department of Biology, 60440-900, Fortaleza, CE, Brazil Federal University of Ceará, Faculty of Pharmacy, Dentistry and Nursing, Department of Pharmacy, 60430-170, Fortaleza, CE, Brazil d Oswaldo Cruz Foundation, Oswaldo Cruz Institute, Laboratory of Structural Biology, 21040-360, Rio de Janeiro, RJ, Brazil b c
A R T I C LE I N FO
A B S T R A C T
Keywords: CXCL10 Leishmania infantum Treg cells
American visceral leishmaniasis is caused by the protozoan Leishmania infantum. The control of the disease depends on the magnitude of the Th1 cell response and IL-10 producing regulatory T cells. Administration of chemokine, such as CXCL10, has shown promising results in the leishmaniasis treatment. Previous studies from our group have shown that CXCL10 induces a reduction in parasite burden in the spleen and a decrease in IL-10 and TGF-β production in L. infantum-infected BALB/c mice. This work investigated whether CXCL10-treatment reduces IL-10 + Treg cell populations (CD4+CD25+Foxp3+ and Tr1) and induces morphological changes in the spleen. BALB/c mice were infected and treated or not with CXCL10 on the 1st, 3rd and 7th days of infection. CXCL10-treatment was able to reduce the parasite load in the spleen in L. infantum-infected BALB/c mice and this decrease in the number of parasites correlated with the decrease in size of this organ in treated animals compared to untreated animals. 7, 23, and 45 days post-treatment (p.t.), the phenotype and frequency of IL10 + Treg cells were evaluated by flow cytometry, and the morphological changes of the spleen were analyzed by optical microscopy. After 7 and 23 days p.t., CXCL10-treated animals showed a significant reduction of CD25Foxp3−IL-10+ (Tr1) cells in the spleen when compared to untreated animals, whereas CD4+CD25+Foxp3+IL10+ Treg cells reduced later at 23rd and 45th days p.t. Furthermore, while untreated animals showed a significant positive correlation between IL-10 production and Tr1 cells, in CXCL10-treated group this correlation was negative. Thus, these findings show that treatment with CXCL10 chemokine in L. infantum-infected BALB/c mice results in suppression of IL10+ Treg (Foxp3+ and Tr1) cells in the spleen, associated with a reduction in parasite load and splenomegaly.
1. Introduction
et al., 2006; Santos et al., 2008). In mice models of VL, the control of the disease depends on the magnitude of the Th1 cell response, which leads to the production of IFN-γ whose pro-inflammatory action is fundamental (Kaye et al., 2004). IL-10 is an immunoregulatory cytokine present in high levels during the disease active stage, and the association of immunosuppression induced by IL-10 in human VL is well established
Visceral leishmaniasis (VL) affects 500,000 people directly and about 350 million people are at risk, including Brazil (Pace, 2014; WHO, 2017) whose disease is caused by L. infantum (Maurício et al., 2000). Treatment of this disease causes, among other problems, toxic effects and resistance of strains to the drugs currently in use (Croft
∗ Corresponding author. Department of Pathology and Legal Medicine, Federal University of Ceará, Alexandre Baraúna Street, 949, Fortaleza, CE, 60430-160, Brazil. E-mail addresses: [email protected] (W.M. Eufrásio de Figueiredo), [email protected] (F.F. Heredia), [email protected] (A.S. Santos), [email protected] (R. da Rocha Braga), [email protected] (F.R. Marciano Fonseca), [email protected] (N. Lúcia de Castro Rodrigues), [email protected] (T.M. Abreu), [email protected] (M. Maria de Lima Pompeu), [email protected] (H.S. Barbosa), [email protected] (M.J. Teixeira).
https://doi.org/10.1016/j.exppara.2019.107789 Received 13 June 2019; Received in revised form 22 October 2019; Accepted 22 October 2019 Available online 24 October 2019 0014-4894/ © 2019 Elsevier Inc. All rights reserved.
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recovered from the animal, the parasites were cultured at 25 °C in N.N.N. medium, containing Schneider's insect medium (Sigma-Aldrich) supplemented with 20% fetal bovine serum (Sigma-Aldrich), 2% sterile human urine and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) (all Sigma-Aldrich). For infection, the parasites were used until the 5th passage in vitro.
(Gupta et al., 2011; Owens et al., 2012; Resende et al., 2013). Regulatory T (Treg) lineage (nTreg and iTreg) is a cell subset regulatory that exhibits forkhead boxP3 (Foxp3) as its transcription factor and regulates Th1 response. The peripheral population of Foxp3+ Treg cells comprises both nTreg and iTreg cells (Curotto de Lafaille and Lafaille, 2009). In addition to the Foxp3+ Treg cells, there are other types of CD4+ Treg cells named Foxp3- type 1 regulatory T (Tr1) cells. These cells are induced in the periphery and have a pivotal role in promoting and maintaining T cell tolerance in BALB/c mice (Yao et al., 2015). In previous studies in Leishmania-infected BALB/c mice, Treg cells presented different roles according to the species of Leishmania used in the infection, contributing either to the pathology and persistence or to resolution of the disease. In the case of L. donovani, evidence indicates that the induction of Treg cells results in an aggravated infection (Martin et al., 2010). In the spleen, the main characteristic of chronic VL is the breakdown of the splenic architecture. In the presence of Leishmania, TNF-α starts to be produced in large quantities triggering production of IL-10 and depletion of the marginal zone (MZ) macrophages (Engwerda et al., 2002; Santana et al., 2008). The deletion of follicular dendritic cells (FDC) and the germinal centers (GC) disorganization prevent the longterm interactions between B cells and T follicular cells necessary for the generation of specific antibodies, that frustrate the action of the parasite (Victora and Nussenzweig, 2012). Exogenous CXCL10 treatment in L. major-infected BALB/c mice has been shown to induce strong recruitment and activation of NK cells (Vester et al., 1999; Muller et al., 2001). Also, treatment with CXCL10 in L. amazonensis-infected BALB/c mice may decrease lesion size and parasitic burden, accompanied by increased production of INF-γ, IL-12 and NO (Vasquez and Soong, 2006). Studies with L. donovani-infected BALB/c mice have demonstrated that CXCL10 is involved in the protective response of infection, inducing a Th1 response, by regulating the pathway of inflammatory mediators such as NO and pro-inflammatory cytokines (Gupta et al., 2009). CXCL10 may also participate in protection against L. donovani with a marked decrease of immunoregulatory cytokines secreted by Treg cells (Gupta et al., 2011). Previous studies of our group have shown an important protective role of CXCL10 in L. infantum-infected BALB/c mice mediated by a significant production of IFN-γ and suppression of the immunoregulatory cytokines IL-10 and TGF-β, leading to the hypothesis if it is not associated with a decrease in the frequency of regulatory T cells (Figueiredo et al., 2017). Therefore, in the present study, we assessed whether treatment with CXCL10 would confer protection against L. infantum infection by regulating the induction of IL10+ Treg cell populations (CD4+CD25+Foxp3+ and Tr1) and also whether treatment with CXCL10 would have repercussions on the changes that occur in the spleen, the organ considered responsible for the persistence of the infection in the visceral leishmaniasis.
2.3. Infection and treatment with CXCL10 Metacyclic promastigotes at the stationary phase were inoculated intraperitoneally (ip.) into BALB/c mice at a concentration of 2 × 107 parasites in 100 μL of sterile saline (NaCl 0.9%). The animals were divided into two groups: one group received CXCL10 (5 μg/kg) (Thermo Scientific) and the other received saline (untreated group) ip. after 1, 3 and 7 days of infection (Gupta et al., 2009). For the histological analysis, an additional uninfected group was used as negative control. The animals were euthanized after 7, 23 and 45 days of treatment by halothane's inhalation (Sigma-Aldrich). For parasitic load analysis, a group of animals was also euthanized on the first day p.t. Six animals per group were euthanized at each time period. The animals were weighed before starting treatment and before euthanasia for weight analysis.
2.4. Parasite load The number of parasites was quantified in the spleen (Titus et al., 1985). The animals were euthanized by inhalation of halothane (SigmaAldrich) and submerged in 3% iodized alcohol up to 3 min to allow decontamination. Then the spleen was removed aseptically, weighed, and one fragment of the spleen (from 0.0671 to 0.0700 g) was homogenized in 1 mL of Schneider medium and left to rest for 5 min. Eight dilutions were made from this cell suspension (10, 50, 100, 500, 103, 104, 105 and 106) in Schneider supplemented with 20% FBS (SigmaAldrich), 2% sterile human urine and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). One hundred microliters of these dilutions were distributed into 96-well plates, flat bottom, 6 wells/dilution. The plates were sealed and incubated at 25 °C for 3 weeks. The wells were observed on an inverted optical microscope (Nikkon) every 3 days to record the dilutions containing promastigotes. The final number of parasites was determined using the ELIDA software (Taswell, 1984).
2.5. Flow cytometry Single cell suspensions were obtained from spleens of animals infected with L. infantum promastigotes, treated or untreated with CXCL10 after seven, 23 and 45 treatment days. For this, each animal spleen was macerated on a nylon filter with pores (70 μm; Falcon) coupled to a 50 mL tube. The obtained cell filtrate was suspended in RPMI medium and subjected to centrifugation (300×g; 5 min; 4 °C). Posteriorly, for regulatory T cell phenotyping, CD4+CD25+Foxp3+IL10+ Treg cells were stained with PerCP Rat Anti-Mouse CD4 (0.2 μg/ test; clone RM4-5), FITC Rat Anti-mouse CD25 (0.5 μg/test; clone 7DA), PE Anti-Mouse/Rat Foxp3 (0.2 μg/test; clone FJK-16s) and APC Rat Anti-Mouse IL-10 (REF: 554468) (all BD Biosciences). For intracellular staining, the cells were permeabilized with a FIX/PERM Kit (eBioscience), according to the manufacturer's guide. For all analyses, the results were compared to those obtained with cells stained with the isotype control antibodies rat IgG2b (A95-1) and rat IgG2a (R35-95) (BD Biosciences, San Diego, CA, USA). After staining, cells were fixed in 1% paraformaldehyde. Cell acquisition was performed using a FACS Calibur flow cytometer and Cell Quest software (BD Biosciences) on at least 30,000 events. Data were plotted and analyzed using Cell Quest (BD Biosciences) and WinMDI 2.9 software (Joseph Trotter, la Jolla, CA, USA).
2. Methods 2.1. Animals Male BALB/c mice, 6–8 weeks old, weighing 18–20g were obtained from the Pathology and Legal Medicine Department of the Federal University of Ceará (DPML/UFC). The animals were housed under specific pathogen-free conditions (12 h light/12 h dark cycle; 22–24 °C), with appropriate commercial feed and water ad libitum. This study was conducted with the Committee on Ethics in the Use of Animals of the UFC approval (CEUA n° 52/2014). 2.2. Parasites The virulence of the L. infantum (MHOM/BR/BA-262) strain was maintained by regular passage in a male golden hamster. After being 2
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parasites in the treated group and this reduction remained after 45 days of treatment (Fig. 1B).
2.6. Histopathological analysis The animals were euthanized by halothane's inhalation in a hermetic chamber, after 7, 23 and 45 treatment days (n = 6), at each time period and from each group). Immediately after euthanasia, the mice were submitted to laparotomy for collecting the spleen. Each spleen was separately weighed to calculate the relative weight (spleen weight/ body weight). The spleens were fixed in 10% buffered formaldehyde, processed and, then, 5 μm microsections of the samples were made and stained with hematoxylin-eosin (H & E). Photomicrographs of 10 fields of the spleen were taken in a 400x magnification to analyze the white pulp. White pulp area and total spleen fragment area were measured digitally by ImageJ® v.1.44 software to analyze the percentage of white pulp.
3.2. Frequency of CD4+CD25+Foxp3+IL-10+ Treg cells in the spleen The gating strategy of Treg (CD4+CD25+Foxp3+) proportion and their expression levels of the cytokine IL-10 can be seen in Fig. 2A, B and 2C. After 7 days of treatment, there was no difference in the frequency of these cells between the CXCL10-treated and untreated animals. However, in the last two post-treatment periods (23 and 45 days) the animals treated with CXCL10 induced a lower frequency of CD4+CD25+Foxp3+IL-10+ Treg cells, compared to untreated animals (day 23, p = 0.0159; day 45, p = 0.0079) (Fig. 2D). In the CXCL10treated animals, the frequency of these cells reached its lowest value (approximately 15%) at day 45 post-treatment, which was equivalent to a reduction of approximately half, when compared to the frequency at day 23 post-treatment that had been 30% (p = 0.0204) (Fig. 2D). On the contrary, in the untreated group, the cell frequency increased between days seven and 23 post-treatment (p = 0.0013), and then it remained steady without any significant decrease between days 23 and 45 post-treatment (Fig. 2D). In addition, a significant correlation between IL-10 production and CD4 +CD25+Foxp3+IL-10+ Treg cells was not observed in the analyzed groups (Fig. 3A and B).
2.7. Statistical analysis The t-Student test was applied to verify the statistical significance between the treated and untreated groups. Comparisons in more than two groups were performed using one-way analysis of variance (ANOVA), followed by Bonferroni post-test. The tests were performed using GraphPad Prism Software version 6.00 (GraphPad Software, San Diego, CA, USA). The results are presented as the arithmetic mean ± mean standard error or as median (whiskers min to max). The minimum significance for all tests was accepted when p < 0.05.
3.3. Frequency of CD4+CD25-Foxp3−IL-10+ Treg (Tr1) cells in the spleen 3. Results
The gating strategy of Treg Tr1 (CD4+CD25-Foxp3-) proportion and their expression levels of the cytokine IL-10 can be seen in Fig. 4A, B and 4C. After 7 treatment days, the frequency of CD4+CD25-Foxp3−IL10+ Tr1 cells in the CXCL10-treated animals was lower when compared to untreated animals (p = 0.0195), and after 23 days of treatment, this frequency remained lower in the CXCL10-treated group (Fig. 4D). After 45 days of treatment, there was no difference in the frequency of these cells between the analyzed groups (Fig. 4D). Farther, CXCL10 treated group showed a significant negative correlation between IL-10 production and Treg Tr1 cells (r2 = 0.518, p = 0.008), while a significant positive correlation was observed in untreated animals (r2 = 0.5109, p = 0.0134) (Fig. 2C and D).
3.1. Relative weight and parasite load in the spleen In the analysis of the relative weight of the spleen was observed that on the 1st day after treatment, the treated group and the control group showed no difference in the weight of the spleen, although the weight of the spleen of both groups (control: 0.0056 ± 0.003; treated: 0.0045 ± 0.0004) was slightly higher than those found in healthy animals (0.0042 ± 0.0002) (Fig. 1A). When the treated group was analyzed separately, there was a decrease in spleen weight of the animals after the 7th day, unlike what happened with the control group, whose weights only declined after 45 days of treatment, and yet were above the weight of the spleen of healthy animals. When the groups were compared, it was observed that there were differences between them on day 7 (p = 0.0286) and 23 (p = 0.0033) after treatment (Fig. 1A). Regarding the number of parasites in the spleen, it was observed that after the 1st day of treatment, animals treated with CXCL10 showed no difference in the number of parasites when compared to control animals (Fig. 1B). After 7 days of treatment, the number of parasites in treated animals remained stable (approximately 104). After 23 days of treatment, there was a decline significant in the number of
3.4. Repercussion of CXCL10 treatment in the spleen CXCL10-treated animals showed a reduced splenomegaly when compared with untreated animals, both at 7 (p = 0.0286) and at 23 days post-treatment (p = 0.0033) (Fig. 5B). However, in relation to the area of white pulp there was no significant difference between the groups non-infected, CXCL10-treated and untreated (Fig. 5A and C and Table 1). Fig. 1. Relative weight (A) and Parasite load (B) in the spleen from BALB/c mice infected by L. infantum and treated with CXCL10. The animals were treated with CXCL10, intraperitoneally (5 μg/kg), or saline at 1, 3 and 7 days of infection. 1, 7, 23 and 45 days after the end of treatment the animals were euthanized and the spleen was removed and used for evaluating the relative weight and parasite load. Data from 4 to 6 animals per group are represented by arithmetic mean ± standard error. Significant differences among groups within the same time period were determined by t-Student test (p < 0.05); differences among the same groups in different time periods were determined by ANOVA (p < 0.05). The dashed lines represent the arithmetic mean of the relative weight of spleen (0.0042) of 6 healthy animals. 3
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Fig. 2. Gating strategy of proportion and frequency of regulatory T cells (CD4+CD25+Foxp3+) and their expression levels of cytokine IL-10 in splenocytes from BALB/c mice infected with L. infantum and treated or untreated with CXCL10. Lymphocytes were identified based on their forward - FSC - and side scatter characteristic - SSC (A). Within the lymphocyte population, CD4+CD25+ cells (B) were gated (R2) and the subpopulation of FoxP3+IL-10+ (C) quantified (R3). Regulatory T cells frequency from at least 6 animals per group are shown as the arithmetic mean ± standard error of the mean, and are representative of 3 independent experiments (D).
Fig. 3. Evaluation of the correlation between IL-10 production and Treg cells. A and B: correlation between IL-10 production and CD4+CD25+Foxp3+IL-10+ Treg cells frequency from untreated and CXCL10-treated BALB/c mice. C and D: correlation between IL-10 production and CD4+CD25-Foxp3−IL-10+ Tr1 cells frequency from untreated and CXCL10-treated BALB/c mice. 4
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Fig. 4. Gating strategy of proportion and frequency of Tr1 cells (CD4+CD25− Foxp3-) and their expression levels of cytokine IL-10 in splenocytes from BALB/c mice infected with L. infantum and treated or untreated with CXCL10. Lymphocytes were identified based on their forward - FSC - and side scatter characteristic - SSC (A). Within the lymphocyte population, CD4+CD25− cells (B) were gated (R2) and the subpopulation of Foxp3−IL-10+ (C) quantified (R3). Tr1 cells frequency from at least 6 animals per group are shown as the arithmetic mean ± standard error of the mean, and are representative of 3 independent experiments (D).
4. Discussion
suggesting a minor role of these cells during visceral L. infantum infection. On the other hand, the same study showed that Tr1 cells were an important source of IL-10, since they were in fact capable of specifically producing large amounts of this cytokine, early on and throughout the experimental period (Rodrigues et al., 2009). Herein, the frequency of Treg Tr1 cells in the CXCL10-treated animals was lower when compared to untreated animals at 7- and 23-days post-treatment. These data suggest that CXCL10 was able to act earlier to regulate this Tr1 regulatory population. Tr1 cells may be found two weeks after infection, producing both IFN-γ and IL-10, and reach a plateau in one month, representing 2–5% of the total splenic CD4+ T cell pool (Owens et al., 2012; Resende et al., 2013). The polarization for this population of CD4+T-bet+IFN-γ+IL-10+ T lymphocytes (Foxp3and CD25−) is associated with increased host susceptibility to Leishmania and initially depends on its activation only by infected dendritic cells known to have low levels of costimulatory molecules on their surfaces and a capacity to produce high amounts of IL-10 (Resende et al., 2013). The persistence of L. infantum in the spleen has a complex immunopathology and is still unknown in its particularities, since it involves several cells of the reticuloendothelial system, cytokines and chemokines. This persistence occurs due to the inability of the host to control the parasite load and to maintain the pro-inflammatory Th1 immune profile; this is due in part to the IL-10 production stimulated by the parasites (Moore et al., 2001). The spleen is a site of active infection and produces several hematological abnormalities in the host, causing the typical symptoms of infection, among them, splenomegaly directly related to the lymphoid follicle hyperplasia of the white pulp (Engwerda et al., 2002; Moore et al., 2001). Visceral leishmaniasis causes changes in the spleen normal physiology through a deep remodeling (Engwerda et al., 2002), with the loss of cellular organization in its intricate microarchitecture. The spleen has several populations of macrophages and structures (germinal centers) that allow antigenic presentation to activate effector cells, T and B lymphocytes (Allen et al., 2007). The MZ macrophages is compromised during infection by L. donovani, generating host immunosuppression (Engwerda et al., 2002; Santana et al., 2008). The next step of our study was to investigate how CXCL10-treatment could affect the spleen. Indeed, CXCL10-treated animals showed a
CXCL10, an IFN-γ-inducing chemokine, is a cytokine that in recent years has been explored in the field of immunotherapy as a potential drug for leishmaniasis. The CXCL10 effects, such as leukocyte activation, balance between regulatory and effector T cells, chemotaxis, hematopoiesis and increased natural killer (NK) cell cytotoxicity (Gupta et al., 2011) may all be of great benefit for the leishmaniasis treatment. Herein, we assessed whether treatment with CXCL10 would confer protection against L. infantum infection by regulating the induction of IL10+ Treg (CD4+CD25+Foxp3+ and Tr1) cell populations and also whether treatment with CXCL10 would have repercussions on the changes that occur in the spleen. In the present study, it was observed that CXCL10-treatment was able to reduce the parasite load in the spleen, four weeks after infection, representing a significant decrease in the number of parasites in this organ. This reduction in the number of parasites in the spleen correlated with the decrease in size of this organ in treated animals compared to control animals. Previous studies have shown that during infection by L. infantum in mice, the parasites multiply rapidly during the first 4 weeks in the liver and disappear spontaneously around the 8th week of infection, whereas in the spleen parasites grow more slowly, leading to a chronic infection in the organ (Wilson et al., 1996). CXCL10 treatment has been shown to induce, in the murine model, a higher production of Th1 cytokines, such as IFN-γ and IL-12, and a reduction of the suppressor cytokines IL-10 and TGF-β levels (Gupta et al., 2009, 2011; Figueiredo et al., 2017). IL-10 and TGF-β are produced by Th2 and Treg cells acting on immunosuppression, which aggravates visceral leishmaniasis (Gupta et al., 2011). In the present study, the CXCL10-treated animals presented the CD4+CD25+Foxp3+ Treg cells producing IL-10 frequency significantly lower in the last two post-treatment periods (23 and 45 days) when compared to the untreated group. These results corroborate with a study which showed that CXCL10 treatment results in protection in vivo against L. donovani, through a significant reduction of the immunosuppressive cytokines IL-10, due to modulation of regulatory T cells (Gupta et al., 2011). Unlike our results, some studies have shown that CD4+CD25+Foxp3+ T cells from L. infantum-infected BALB/c mice were able to secrete some IL-10 levels but nothing very expressive, 5
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Fig. 5. Repercussion of CXCL10 treatment in the spleen. White pulp area (A), spleen weight index (B), and spleen histological sections (C) of BALB/c mice infected with L. infantum treated or untreated with CXCL10; and non-infected. The animals were treated with CXCL10 (5 μg/kg) or sterile saline, ip., on the 1st, 3rd and 7th days of infection. After 7, 23 and 45 days of treatment, the animals were euthanized to collect the spleen. Data from at least 6 animals per group are shown as median (whiskers min to max) and are representative of 2 independent experiments. Histological sections: original magnification ×400.
inoculum is killed by MZ macrophages within the first 24 h after infection (Allen et al., 2007). CXCL10 induces IFN-γ secretion (Gupta et al., 2009, 2011; Figueiredo et al., 2017), produced by NK cells, which is an important cytokine that induces macrophage activation (Alexander, 2005; Dey et al., 2007). Other studies have pointed to the increased cytotoxicity of NK cells in BALB/c mice infected with L. major and treated with CXCL10 (Dey et al., 2007), which suggest that this cytotoxicity could help maintain the spleen structure by eliminating infected macrophages.
Table 1 White pulp area in relation to total spleen area (%) in BALB/c mice infected with L. infantum and treated with CXCL10. Time (days post-treatment)
7 23 45
Infected CXCL10
UNTREATED
30.3 24.9 37.0
36.5 27.7 38.4
P
ns ns ns
ns: no significant.
5. Conclusion reduced splenomegaly when compared with untreated animals. However, in relation to the area of white pulp there was no significant difference between the groups. About 50% of the initial parasite
In conclusion, the findings of this study, associated with a lower spleen weight and a reduction the parasite load in the spleen in the CXCL10-treated animals, suggests a protective role of CXCL10 6
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chemokine with suppression of IL10+ Treg (Foxp3+ and Tr1) cells in the spleen in L. infantum-infected BALB/c mice. These data may provide information for the development of future therapeutic interventions, using CXCL10 as an adjuvant in the visceral leishmaniasis.
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Funding This work was supported by Brazilian grants from Conselho Nacional de Pesquisa (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and CAPES-Programa DINTER (Doutorado Interinstitucional) Medicina Tropical 309/2013. Availability of data and materials All data generated or analyzed during this study are included in this published article. Declarations of competing interest None. Acknowledgements The authors wish to thank Dr. Andrea Henrique Pons of the Laboratory of Innovations in Therapies, Education and Bioproducts, Oswaldo Cruz Institute, and Dr. Adriano Gomes da Silva of the Evandro Chagas National Institute of Infectology, for their scientific advice on Treg cells and flow cytometry. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.exppara.2019.107789. References Alexander, J., 2005. Bryson K. T helper (h)1/Th2 and Leishmania: paradox rather than paradigm. Immunol. Lett. 99 (1), 17–23. Allen, C.D., Okada, T., Cyster, J.G., 2007. Germinal-center organization and cellular dynamics. Immunity 27 (2), 190–202. Croft, S.L., Sundar, S., Fairlamb, A.H., 2006. Drug resistance in leishmaniasis. Clin. Microbiol. Rev. 19 (1), 111–126. Curotto de Lafaille, M.A., Lafaille, J.J., 2009. Natural and adaptive foxp3+ regulatory T cells: more of the same or a division of labor? Immunity 30 (5), 626–635. Dey, R., Majumder, N., Bhattacharyya, M.S., Bhattacharjee, S., Banerjee, S., Roy, S., et al., 2007. Induction of host protective Th1 immune response by chemokines in Leishmania donovani-infected BALB/c mice. Scand. J. Immunol. 66 (6), 671–683. Engwerda, C.R., Ato, M., Cotterell, S.E., Mynott, T.L., Tschannerl, A., Gorak-Stolinska, P.M., et al., 2002. A role for tumor necrosis factor-alpha in remodeling the splenic marginal zone during Leishmania donovani infection. Am. J. Pathol. 161 (2), 429–437. Figueiredo, W.M.E., Viana, S.M., Alves, D.T., Guerra, P.V., Coêlho, Z.C.B., Barbosa, H.S.,
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