Deletion of IL-4 Receptor Alpha on Dendritic Cells Renders BALB/c Mice Hypersusceptible to Leishmania major Infection

In BALB/c mice, susceptibility to infection with the intracellular parasite Leishmania major is driven largely by the development of T helper 2 (Th2) responses and the production of interleukin (IL)-4 and IL-13, which share a common receptor subunit, the IL-4 receptor alpha chain (IL-4Rα). While IL-4 is the main inducer of Th2 responses, paradoxically, it has been shown that exogenously administered IL-4 can promote dendritic cell (DC) IL-12 production and enhance Th1 development if given early during infection. To further investigate the relevance of biological quantities of IL-4 acting on DCs during in vivo infection, DC specific IL-4Rα deficient (CD11c^creIL-4Rα^-/lox) BALB/c mice were generated by gene targeting and site-specific recombination using the cre/loxP system under control of the cd11c locus. DNA, protein, and functional characterization showed abrogated IL-4Rα expression on dendritic cells and alveolar macrophages in CD11c^creIL-4Rα^-/lox mice. Following infection with L. major, CD11c^creIL-4Rα^-/lox mice became hypersusceptible to disease, presenting earlier and increased footpad swelling, necrosis and parasite burdens, upregulated Th2 cytokine responses and increased type 2 antibody production as well as impaired classical activation of macrophages. Hypersusceptibility in CD11c^creIL-4Rα^-/lox mice was accompanied by a striking increase in parasite burdens in peripheral organs such as the spleen, liver, and even the brain. DCs showed increased parasite loads in CD11c^creIL-4Rα^-/lox mice and reduced iNOS production. IL-4Rα-deficient DCs produced reduced IL-12 but increased IL-10 due to impaired DC instruction, with increased mRNA expression of IL-23p19 and activin A, cytokines previously implicated in promoting Th2 responses. Together, these data demonstrate that abrogation of IL-4Rα signaling on DCs is severely detrimental to the host, leading to rapid disease progression, and increased survival of parasites in infected DCs due to reduced killing effector functions.     

IL-4- and IL-4 receptor-deficient BALB/c mice reveal differences in susceptibility to Leishmania major parasite substrains

Using genetically pure BALB/c mice deficient in IL-4 (IL-4-/-) or IL-4 receptor alpha-chain (IL-4Ralpha-/-), we have observed different disease outcomes to Leishmania major infection depending on the parasite substrain. Infection with L. major LV39 caused progressive, nonhealing ulcers and uncontrolled parasite growth in both IL-4-/- and IL-4Ralpha-/- mice. In contrast, infection with L. major IR173 was partially controlled in IL-4-/- mice but efficiently controlled in IL-4Ralpha-/- mice. Both IL-4-/- and IL-4Ralpha-/- mice infected with either substrain displayed reduced Th2 responses. Surprisingly, IFN-gamma secretion was not up-regulated in the mutant mice, even in the IL-4Ralpha-/- mice, which were resistant to L. major IR173. The lack of increased IFN-gamma production suggests that cytokine cross-regulation may not be operating in this model and that the effective ratios of Th1/Th2 cytokines become more indicative of disease outcome. The partial vs complete resistance to IR173 in IL-4-/- or IL-4Ralpha-/- mice implies that, in addition to IL-4, IL-13 may be involved in disease progression during L. major infection. The results with LV39 infection indicate that yet another unidentified factor is capable of causing susceptibility to L. major in the absence of IL-4 or IL-4 signaling.     

Despite increased CD4+Foxp3+ cells within the infection site, BALB/c IL-4 receptor-deficient mice reveal CD4+Foxp3-negative T cells as a source of IL-10 in Leishmania major susceptibility

BALB/c IL-4Ralpha(-/-) mice, despite the absence of IL-4/IL-13 signaling and potent Th2 responses, remain highly susceptible to Leishmania major substain LV39 due exclusively to residual levels of IL-10. To address the contribution of CD4(+)CD25(+) T regulatory (Treg) cells to IL-10-mediated susceptibility, we depleted CD4(+)CD25(+) cells in vivo and reconstituted IL-4Ralpha x RAG2 recipients with purified CD4(+)CD25(-) T cells. Although anti-CD25 mAb treatment significantly decreased parasite numbers in IL-4Ralpha(-/-) mice, treatment with anti-IL-10R mAb virtually eliminated L. major parasites in both footpad and dermal infection sites. In addition, IL-4Ralpha x RAG2 mice reconstituted with CD4(+) cells depleted of CD25(+) Treg cells remained highly susceptible to infection. Analysis of L. major-infected BALB/c and IL-4Ralpha(-/-) inflammatory sites revealed that the majority of IL-10 was secreted by the CD4(+)Foxp3(-) population, with a fraction of IL-10 coming from CD4(+)Foxp3(+) Treg cells. All T cell IFN-gamma production was also derived from the CD4(+)Foxp3(-) population. Nevertheless, the IL-4Ralpha(-/-)-infected ear dermis, but not draining lymph nodes, consistently displayed 1.5- to 2-fold greater percentages of CD4(+)CD25(+) and CD4(+)Foxp3(+) Treg cells compared with the BALB/c-infected dermis. Thus, CD4(+)Foxp3(-) T cells are a major source of IL-10 that disrupts IFN-gamma activity in L. major-susceptible BALB/c mice. However, the increase in CD4(+)Foxp3(+) T cells within the IL-4Ralpha(-/-) dermis implies a possible IL-10-independent role for Treg cells within the infection site, and may indicate a novel immune escape mechanism used by L. major parasites in the absence of IL-4/IL-13 signaling.     

Protective immunity against Leishmania major induced by Leishmania tropica infection of BALB/c mice

Leishmania (L.) tropica is a causative agent of human cutaneous and viscerotropic leishmaniasis. Immune response to L. tropica in humans and experimental animals are not well understood. We previously established that L. tropica infection induces partial protective immunity against subsequent challenge infection with Leishmania major in BALB/c mice. Aim of the present study was to study immunologic mechanisms of protective immunity induced by L. tropica infection, as a live parasite vaccine, in BALB/c mouse model. Mice were infected by L. tropica, and after establishment of the infection, they were challenged by L. major. Our findings shows that L. tropica infection resulted in protection against L. major challenge in BALB/c mice and this protective immunity is associated with: (1) a DTH response, (2) higher IFN-γ and lower IL-10 response at one week post-challenge, (3) lower percentage of CD4⁺ lymphocyte at one month post-challenge, and (4) the source of IFN-γ and IL-10 were mainly CD4⁻ lymphocyte up to one month post-challenge suggesting that CD4⁻ lymphocytes may be responsible for protection induced by L. tropica infection in the studied intervals.     

The opioid antagonist naloxone inhibits Leishmania major infection in BALB/c mice

BALB/c mice are susceptible to develop non-healing, progressive infection with Leishmania major (L. major) due to the development of a non-protective Th2 response. Resistance to L. major infection is dependent to Th1 response. Treatment of mice with the opioid antagonist naloxone can promote the activation of Th1 responses. Here we study the effect of chronic administration of various doses of naloxone on susceptibility of BALB/c mice to L. major infection. Our results showed that naloxone has dose-dependent biphasic effect on L. major infection in BALB/c mice. While administration of 1mg/kg × 2/day tends to exacerbate the local reaction to L. major infection, treatment with 10mg/kg × 2/day of naloxone suppresses the local reaction and progress of infection. On the other hand treatment of mice with middle dose (5mg/kg whether 1 or 2 times per day) does not have significant effect on the infection. This study demonstrates that administration of high dose of naloxone could improve protection against L. major infection in BALB/c mice, presumably by modulation in Th1/Th2 balance or by affecting macrophages through binding to Toll-like receptors.     

Influence of CD4+CD25+ T cells on Plasmodium berghei NK65 infection in BALB/c mice

CD4(+) T cells co-expressing CD25 (CD4(+)CD25(+) T cells) have been identified as immunoregulatory suppressors modulating autoimmune response. Beside that, autoimmune response was supposed to be associated with malaria infection. Based on these data, we hypothesised that CD4(+)CD25(+) T cells may influence protective immunity to malaria parasites, while suppressing autoimmune response arising throughout the course of malarial infection. To test this possibility, we evaluated the kinetics of CD4(+)CD25(+) T cells during malaria infection and investigated the influence of CD25 depletion by anti-mouse CD25 monoclonal antibody (PC61) on the infection, using a mouse model of premunition to Plasmodium berghei NK65 malaria. The results showed that, during exacerbation of P. berghei NK65 infection, the proportion of CD4(+)CD25(+) T cells among CD4(+) T cells decreased, although that of CD4(+) T cells increased. CD25 depletion clearly delayed the growth of parasitaemia during parasite challenge, particularly in immunised mice. These findings demonstrated that CD4(+)CD25(+) T cells are able to influence protective immunity underlying premunition to P. berghei NK65 parasites.     

Higher frequency of Leishmania major-specific L3T4+ T cells in susceptible BALB/c as compared with resistant CBA mice

In previous studies, we reported that a) the adoptive transfer of parasite-specific L3T4+ T cells enhanced rather than inhibited the development of lesions induced by Leishmania major in normal BALB/c mice, and b) the depletion in vivo of L3T4+ T cells by administration of anti-L3T4 monoclonal antibody reversed the susceptibility of BALB/c mice to L. major. To further assess the role of specific L3T4+ T cells in the development of lesions induced by L. major in BALB/c mice, the frequency of parasite-specific T cells capable of mediating specific delayed-type hypersensitivity (DTH) reactivity was determined, by limiting dilution analysis, in the lymph nodes draining the lesions of susceptible (BALB/c) and resistant (CBA) mice. The numbers of L. major-specific DTH-mediating T cells was found to be substantially increased in the lymph nodes of infected BALB/c mice as compared with CBA mice. Moreover in CBA mice, analysis of the cell surface phenotype of the L. major-specific DTH-mediating T cells showed that these cells were equally represented in the L3T4+, Lyt-2-, and L3T4- Lyt-2+ subsets, whereas the majority of these cells in BALB/c mice expressed the L3T4+ Lyt-2- surface phenotype.     

The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+CD25+ T cells

Susceptibility and development of Th2 cells in BALB/c mice infected with Leishmania major result from early IL-4 production by Vbeta4Valpha8 CD4+ T cells in response to the Leishmania homolog of mammalian RACK1 Ag. A role for CD4+CD25+ regulatory T cells in the control of this early IL-4 production was investigated by depleting in vivo this regulatory T cell population. Depletion induced an increase in the early burst of IL-4 mRNA in the draining lymph nodes of BALB/c mice, and exacerbated the course of disease with higher levels of IL-4 mRNA and protein in their lymph nodes. We further showed that transfer of 10(7) BALB/c spleen cells that were depleted of CD4+CD25+ regulatory T cells rendered SCID mice susceptible to infection and allowed Th2 differentiation while SCID mice reconstituted with 10(7) control BALB/c spleen cells were resistant to infection with L. major and developed a Th1 response. Treatment with a mAb against IL-4 upon infection with L. major in SCID mice reconstituted with CD25-depleted spleen cells prevented the development of Th2 polarization and rendered them resistant to infection. These results demonstrate that CD4+CD25+ regulatory T cells play a role in regulating the early IL-4 mRNA and the subsequent development of a Th2 response in this model of infection.     

Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in BALB/c mice

Leishmania tropica and L. major are etiologic agents of human cutaneous leishmaniasis. Delayed type hypersensitivity (DTH) is an immunologic response that has been frequently used as a correlate for protection against or sensitization to leishmania antigen. In BALB/c mice, L. tropica infection results in non-ulcerating disease, whereas L. major infection results in destructive lesions. In order to clarify the immunologic mechanisms of these 2 different outcomes, we compared the ability of these 2 leishmania species in induction of DTH response in this murine model. BALB/c mice were infected with L. major or L. tropica, and disease evolution and DTH responses were determined. The results show that the primary L. major infection can exacerbate the secondary L. major infection and is associated with DTH response. Higher doses of the primary L. major infection result in more disease exacerbation of the secondary L. major infection as well as higher DTH response. L. tropica infection induces lower DTH responses than L. major. We have previously reported that the primary L. tropica infection induces partial protection against the secondary L. major infection in BALB/c mice. Induction of lower DTH response by L. tropica suggests that the protection induced against L. major by prior L. tropica infection may be due to suppression of DTH response.     

Dendritic cell-mediated vaccination relies on interleukin-4 receptor signaling to avoid tissue damage after Leishmania major infection of BALB/c mice

Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11c^creIL-4Rα^−/lox) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×10⁵ stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11c^creIL-4Rα^−/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.     

BALB/c mice bearing a transgenic IL-12 receptor beta 2 gene exhibit a nonhealing phenotype to Leishmania major infection despite intact IL-12 signaling

In BALB/c mice infected with Leishmania major, early secretion of IL-4 leads to a Th2-type response and nonhealing. We explored the role of IL-4-induced down-regulation of the IL-12Rbeta2 chain in the establishment of this Th2 response. First, we showed that the draining lymph nodes of resistant C57BL/6 mice infected with L. major were enriched in CD4+/IL-12Rbeta2 chain+ cells producing IFN-gamma. Next, we demonstrated that BALB/c background mice bearing an IL-12Rbeta2-chain transgene manifested a nonhealing phenotype similar to wild-type littermates despite the persistence of their ability to undergo STAT4 activation. Finally, we found that such transgenic mice display more severe infection than wild-type littermates when treated with IL-12 7 days after infection, and under this condition, the mice display increased Leishmania Ag-induced IL-4 secretion. These studies indicate that although CD4+/IL-12Rbeta2 chain+ T cells are important components of the Th1 response, maintenance of IL-12Rbeta2 chain expression is not sufficient to change a Th2 response to a Th1 response in vivo and thus to allow BALB/c mice to heal L. major infection.     

Antigenic extracts of Leishmania braziliensis and Leishmania amazonensis associated with saponin partially protects BALB/c mice against Leishmania chagasi infection by suppressing IL-10 and IL-4 production

This study evaluated two vaccine candidates for their effectiveness in protecting BALB/c mice against Leishmania chagasi infection. These immunogenic preparations were composed of Leishmania amazonensis or Leishmania braziliensis antigenic extracts in association with saponin adjuvant. Mice were given three subcutaneous doses of one of these vaccine candidates weekly for three weeks and four weeks later challenged with promastigotes of L. chagasi by intravenous injection. We observed that both vaccine candidates induced a significant reduction in the parasite load of the liver, while the L. amazonensis antigenic extract also stimulated a reduction in spleen parasite load. This protection was associated with a suppression of both interleukin (IL)-10 and IL-4 cytokines by spleen cells in response to L. chagasi antigen. No change was detected in the production of IFN-γ. Our data show that these immunogenic preparations reduce the type 2 immune response leading to the control of parasite replication.     

Resolution of an infection with Leishmania braziliensis confers complete protection to a subsequent challenge with Leishmania major in BALB/c mice

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-gamma (IFN-gamma) but low amounts of interleukin-4. The IFN-gamma produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-gamma abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.     

Partial depletion of CD4(+)CD25(+)Foxp3(+) T regulatory cells significantly increases morbidity during acute phase Toxoplasma gondii infection in resistant BALB/c mice

CD4(+)CD25(+)Foxp3(+) T regulatory (Treg) cells, are known to regulate responses to infectious agents. Here we compared disease progression in BALB/c and C57BL/6(B6) mice infected perorally with Toxoplasma gondii for 7 days and examined the affect of partial depletion of Treg cells in these mice. BALB/c mice were seen to be resistant to peroral infection whereas B6 mice were susceptible in terms of mortality. Although the depletion of Treg cells before infection had no effect on the survival of B6 or BALB/c mice, it resulted in increased parasite burdens in BALB/c mice, especially in the lamina propria, but not in B6 mice. Pro-inflammatory cytokines were also increased in Treg cells depleted BALB/c mice as compared to B6 mice. In addition Treg cell depleted BALB/c mice displayed increased ileal histopathology compared to their non-treated counterparts. These findings provide evidence for the contribution of Treg cells, in the resistance of BALB/c mice against peroral T.?gondii infection.     

Immune response to Leishmania (Leishmania) chagasi infection is reduced in malnourished BALB/c mice

Protein-energy malnutrition and micronutrient deficiencies may down-regulate immune response and increase morbidity and mortality due to infection. In this study, a murine model was used to study the effects of protein, iron and zinc deficiencies on the immune response to Leishmania (Leishmania) chagasi infection. Mice were initially fed a standard diet or with a diet containing 3% casein but deficient in zinc and iron. After malnutrition was established, mice were inoculated with L. chagasi and sacrificed four weeks later in order to evaluate liver and spleen parasite loads and serum biochemical parameters. Significant decreases in liver and spleen weight, an increase in the parasite loads in these organs and decreases in serum protein and glucose concentrations in malnourished animals were observed. Furthermore, the production of interferon-gamma by spleen cells from infected malnourished mice stimulated by Leishmania antigen was significantly lower compared with that in control diet mice. These data suggest that malnutrition alters the immune response to L. chagasi infection in the BALB/c model and, in association with the effects on biochemical and anatomical parameters of the host, favored increases in the parasite loads in the spleens and livers of these animals.     

CD4+CD25+ T regulatory cells limit effector T cells and favor the progression of brucellosis in BALB/c mice

Brucellosis is one of the most common bacterial zoonoses worldwide. Infection is usually chronic and sometimes lifelong. Different mechanisms can be postulated as to the basis for the induction of the chronic status of brucellosis, but a comprehensive knowledge is still lacking. Here, we carried out a series of experiments in order to assess if the persistence of Brucella abortus could be ascribed to the effect of a down regulation of the immune response due to activity of regulatory T cells. We demonstrate that CD4 + CD25 + T regulatory cells are able to limit the effectiveness of CD4 + T cells and are able to favor the maintenance and the progression of B. abortus infection.     

Role of interleukin-4 and prostaglandin E2 in Leishmania amazonensis infection of BALB/c mice

The role of cytokines in Leishmania amazonensis experimental infection has not been as well studied as in Leishmania major infection model. Here we investigated the role of interleukin (IL)-4 and PGE(2) in L. amazonensis infection of susceptible BALB/c mice. IL-4 deficient (-/-) or wild-type (+/+) BALB/c mice were infected with different inocula of L. amazonensis. Two weeks after infection with 5x10(6) promastigotes/footpad, the production of interferon (IFN)-gamma upon L. amazonensis antigen stimulation was significantly higher in lymph node cell cultures of IL-4-/- mice than in IL-4+/+ mice. The levels of anti-leishmania IgG2a antibodies were also significantly higher in serum from IL-4-/- mice. In contrast, the levels of IgG1 antibodies were increased in IL-4+/+ mice and almost undetectable in IL-4-/- mice. Despite the increased Th1 response, lesions of IL-4-/- BALB/c mice progressed similarly to those of IL-4+/+ mice upon infection with the 5x10(6) inoculum. However, IL-4-/- mice developed smaller lesions upon infection with 10(5), 10(4) or 10(3) parasites than IL-4+/+ mice. The resistance of IL-4-/- correlated with higher Th1 response, compared to IL-4+/+ upon infection with 10(4)L. amazonensis. IL-4+/+ mice treated with indomethacin, an inhibitor of PGE(2) synthesis, during the first 3weeks of infection developed smaller lesions and lower parasitic load when compared to the control group. The lesions of indomethacin-treated groups contained mostly macrophages without vacuoles and small or absent necrotic areas. These results indicate that IL-4 and PGE(2) are susceptibility factors to L. amazonensis infection.     

The central memory CD4+ T cell population generated during Leishmania major infection requires IL-12 to produce IFN-gamma

Central memory CD4(+) T cells provide a pool of lymph node-homing, Ag-experienced cells that are capable of responding rapidly after a secondary infection. We have previously described a population of central memory CD4(+) T cells in Leishmania major-infected mice that were capable of mediating immunity to a secondary infection. In this study, we show that the Leishmania-specific central memory CD4(+) T cells require IL-12 to produce IFN-gamma, demonstrating that this population needs additional signals to develop into Th1 cells. In contrast, effector cells isolated from immune mice produced IFN-gamma in vitro or in vivo in the absence of IL-12. In addition, we found that when central memory CD4(+) T cells were adoptively transferred into IL-12-deficient hosts, many of the cells became IL-4 producers. These studies indicate that the central memory CD4(+) T cell population generated during L. major infection is capable of developing into either Th1 or Th2 effectors. Thus, continued IL-12 production may be required to ensure the development of Th1 cells from this central memory T cell pool, a finding that has direct relevance to the design of vaccines dependent upon central memory CD4(+) T cells.     

BALB/c mice infected with antimony treatment refractory isolate of Leishmania braziliensis present severe lesions due to IL-4 production

Background

Leishmania braziliensis is the main causative agent of cutaneous leishmaniasis in Brazil. Protection against infection is related to development of Th1 responses, but the mechanisms that mediate susceptibility are still poorly understood. Murine models have been the most important tools in understanding the immunopathogenesis of L. major infection and have shown that Th2 responses favor parasite survival. In contrast, L. braziliensis–infected mice develop strong Th1 responses and easily resolve the infection, thus making the study of factors affecting susceptibility to this parasite difficult.

Methodology/Principal Findings

Here, we describe an experimental model for the evaluation of the mechanisms mediating susceptibility to L. braziliensis infection. BALB/c mice were inoculated with stationary phase promastigotes of L. braziliensis, isolates LTCP393(R) and LTCP15171(S), which are resistant and susceptible to antimony and nitric oxide (NO), respectively. Mice inoculated with LTCP393(R) presented larger lesions that healed more slowly and contained higher parasite loads than lesions caused by LTCP15171(S). Inflammatory infiltrates in the lesions and production of IFN-γ, TNF-α, IL-10 and TGF-β were similar in mice inoculated with either isolate, indicating that these factors did not contribute to the different disease manifestations observed. In contrast, IL-4 production was strongly increased in LTCP393(R)-inoculated animals and also arginase I (Arg I) expression. Moreover, anti-IL-4 monoclonal antibody (mAb) treatment resulted in decreased lesion thickness and parasite burden in animals inoculated with LTCP393(R), but not in those inoculated with LTCP15171(S).

Conclusion/Significance

We conclude that the ability of L. braziliensis isolates to induce Th2 responses affects the susceptibility to infection with these isolates and contributes to the increased virulence and severity of disease associated with them. Since these data reflect what happens in human infection, this model could be useful to study the pathogenesis of the L. braziliensis infection, as well as to design new strategies of therapeutic intervention.