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.     

Interleukin-18 enhances a Th2 biased response and susceptibility to Leishmania mexicana in BALB/c mice

Interleukin-18 deficient mice on a BALB/c background display increased resistance to cutaneous infection with Leishmania mexicana, with reduced lesion progression and reduced parasite burdens compared with wild-type mice. Infected IL-18-/- mice had lower antigen specific IgG1 levels and total IgE levels and conversely higher antigen specific IgG2a levels than similarly infected wild-type mice. Splenocytes isolated from infected IL-18-/- mice produced significantly lower levels of antigen induced IL-4 and higher levels of IFN-gamma than wild-type animals. Consequently IL-18 during L. mexicana infection of BALB/c mice promotes a Th2 biased response and thereby has a disease exacerbating role.     

Lymph node cell responsiveness in BALB/c mice infected with Leishmania mexicana

In the present study we measured the blastogenic response of lymph node cells from BALB/c mice infected with Leishmania mexicana throughout the course of infection. Results showed that infected mice displayed normal blastogenic responses in the lymph nodes until twenty weeks of infection. Thereafter, there was a gradual suppression. Comparison of the immunoresponsiveness in the spleen and lymph nodes, revealed normal responses in the lymph nodes several weeks after suppression in the spleen had occurred. Suppression of blastogenic responses in the lymph nodes was related to an adherent macrophage-like cell which actively suppressed normal proliferative responses to mitogens.     

Protective effect of lectin from Synadenium carinatum on Leishmania amazonensis infection in BALB/c mice

The protective effect of the Synadenium carinatum latex lectin (ScLL), and the possibility of using it as an adjuvant in murine model of vaccination against American cutaneous leishmaniasis, were evaluated. BALB/c mice were immunized with the lectin ScLL (10, 50, 100 microgram/animal) separately or in association with the soluble Leishmania amazonensis antigen (SLA). After a challenge infection with 10(6) promastigotes, the injury progression was monitored weekly by measuring the footpad swelling for 10 weeks. ScLL appeared to be capable of conferring partial protection to the animals, being most evident when ScLL was used in concentrations of 50 and 100 microgram/animal. Also the parasite load in the interior of macrophages showed significant reduction (61.7%) when compared to the control group. With regard to the cellular response, ScLL 50 and 100 microgram/animal stimulated the delayed-type hypersensitivity (DTH) reaction significantly (P < 0.05) higher than SLA or SLA plus ScLL 10 weeks after the challenge infection. The detection of high levels of IgG2a and the expression of mRNA cytokines, such as IFN-gamma, IL-12, and TNF-alpha (Th1 profiles), corroborated the protective role of this lectin against cutaneous leishmaniasis. This is the first report of the ScLL effect on leishmaniasis and shows a promising role for ScLL to be explored in other experimental models for treatment of leishmaniasis.     

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.     

Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge

Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccination using Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasite species. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver and spleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathology and a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production of IFN-γ by CD4⁺ and CD8⁺ T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies were detected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.     

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.     

Leishmania amazonensis infection: a comparison of in vivo leishmanicidal mechanisms between immunized and naive infected BALB/c mice.

In vitro studies have shown that both macrophage activation and destruction of parasitized macrophages lead to leishmania destruction. The relative role played by such mechanisms in vivo have not been properly evaluated. We took advantage of the model of intravenous immunization with solubilized leishmanial antigen which renders partially resistant the otherwise highly susceptible BALB/c mice to address this issue avoiding the interference of different genetic backgrounds. Leishmania destruction occurred in three situations: destruction of the parasitized macrophage, which were in close contact with lymphocytes or eosinophils; extracellular damage, always surrounded by small foci of granulocytes; and parasite damage inside activated macrophages. Destruction of the parasitized macrophages was frequently seen in immunized and protected animals. Our observations suggest that destruction of parasite-loaded macrophages is an important mechanism of host protection in experimental cutaneous leishmaniasis.     

Down-regulation of gp63 level in Leishmania amazonensis promastigotes reduces their infectivity in BALB/c mice

Episomal expression of the major surface glycoprotein (gp63) sense and antisense mRNAs in Leishmania amazonensis was found previously to modulate the expression of this molecule as well as its infection of macrophages in vitro. Here, we evaluated the in vivo infectivity of these transfectants in BALB/c mice. Antisense downregulation of gp63 renders this parasite sensitive to complement-mediated lysis and less infective to mice, as indicated by a delay in lesion development and a significant reduction in lesion size and parasite loads at the site of inoculation and in the draining lymph nodes (DLNs). CD4+ cells at the site of inoculation decreased in number more rapidly and were 2-fold less numerous than those in controls by week 4. The number of IFN-gamma-positive cells was higher, while IL-10 positive cells were undetectable. In DLNs, CD4+ cells were higher in number, and the profile of cytokine-positive cells followed essentially the same patterns--found at the site of inoculation. These results suggest that the downregulation of gp63 increases extracellular lysis of the mutants by complement, in the in vivo environment, and reduces their infection of macrophages, resulting in a type 1 immune response seen at the site of inoculation and DLNs.     

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.     

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.     

Leishmania amazonensis infection in nude mice.

Leishmania amazonensis is an intracellular protozoan parasite of macrophages. Cutaneous leishmaniasis in an immunocompetent host begins as papules or nodules followed by ulceration at the site of promastigote inoculation. In this study, the pathological changes of cutaneous leishmaniasis lesions in T cell deficient nude mice were examined. When infected with L. amazonensis promastigotes, nude mice developed non-ulcerative cutaneous nodules. By histological examination of cutaneous lesions, massive accumulation of vacuolated histiocytes containing amastigotes was observed in all the nude mice. Although infiltration of mononuclear and polymorphonuclear cells was seen in the lesions of immunocompetent mice, few such cells were observed in the lesions of nude mice. These results indicate the importance of T cells on the ulcer formation in cutaneous leishmaniasis.     

Some of the early events underlying Th2 cell maturation and susceptibility to Leishmania major infection in BALB/c mice.

The first experimental evidence for the development of polarized CD4+ Th1 and Th2 responses in vivo has been obtained using the murine model of infection with Leishmania major, an intracellular parasite of macrophages in their vertebrate host. Genetically determined resistance and susceptibility to infection with this parasite have been clearly demonstrated to result from the development of polarized Th1 and Th2 responses, respectively. Using this model system, the dominant role of cytokines in the induction of polarized CD4+ responses has been validated in vivo. The requisite role of IL-4 in mediating both Th2 differentiation and susceptibility to infection in BALB/c mice has directed interest towards the search for evidence of IL-4 production early after infection and identification of its cellular source. We have been able to demonstrate a burst of IL-4 production in susceptible BALB/c mice within the first day of infection with L. major and could establish that this rapidly produced IL-4 instructed Th2 lineage commitment of subsequently activated CD4+ T cells and stabilized this commitment by downregulating IL-12 Rbeta2 chain expression, resulting in susceptibility to infection. Strikingly, this early IL-4 response to infection resulted from the cognate recognition of a single epitope in a distinctive antigen, LACK, from this complex microorganism by a restricted population of CD4+ T cells that express Vbeta4-Valpha8 T cell receptors.     

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.     

The surface free energy of Leishmania mexicana amazonensis.

Surface charge of Leishmania mexicana amazonensis was investigated by direct zeta-potential determination and ultrastructural cytochemistry, and its surface tension was studied by measurements of the advancing contact angle formed by the parasite monolayers with drops of liquids of different polarities. Both virulent and avirulent promastigotes exhibited negatively charged surfaces with a zeta-potential of about -15 mV. Treatment of these cells with trypsin, alkaline phosphatase, or phospholipase C rendered their surfaces less negatively charged, whereas neuraminidase did not alter the parasite negativeness. Cytochemically, we could observe a reduction in the cationized ferritin binding after the parasite treatment with each of the former enzymes, but not with neuraminidase. The surface free energy of parasites was calculated by taken to account the London dispersion, the Keeson dipole-dipole, and the Debye dipole-induced forces, as well as the surface polarity of the parasites and their zeta-potentials, by considering their adhesion to polystyrene surfaces. The delta G values of -6.4 and -18.1 mJ.m-2 were obtained for avirulent and virulent promstigotes, respectively.     

Inhibition of growth of Leishmania mexicana mexicana by Leishmania mexicana amazonensis during "in vitro" co-cultivation

Inhibition of one Leishmania subspecies by exometabolites of another subspecies, a phenomenon not previously reported, is suggested by our recent observations in cell cloning experiments with Leishmania mexicana mexicana and Leishmania mexicana amazonensis. Clones were identified using the technique of schizodeme analysis. The phenomenon observed is clearly relevant to studies of parasite isolation, leishmanial metabolism, cross-immunity and chemotherapy.     

Interferon-gamma and interleukin-12 mediate protection to acute Neospora caninum infection in BALB/c mice

The type of immune response required to protect mice against clinical disease during acute Neospora caninum challenge was investigated in BALB/c mice. Groups of female BALB/c mice were infected i.p. with N. caninum tachyzoites concomitant with either: (1) antibody to interferon-gamma; (2) recombinant murine interleukin-12; or (3) recombinant murine interleukin-12 plus antibody to interferon-gamma. Mice treated with anti-interferon-gamma alone had increased morbidity/mortality, decreased body weight, increased foci of liver necrosis and increased numbers of N. caninum tachyzoites in the lung by 7 days p.i. compared with controls. Increased disease and parasite load in the anti-interferon-gamma-treated mice was associated with antigen-specific antibody IgG1 > IgG2a and a three-fold decreased ratio of antigen-specific interferon-gamma:interleukin-4. Mice treated with recombinant murine interleukin-12 had decreased encephalitis and brain parasite load at 3 weeks p.i. compared with control mice treated with PBS. In recombinant murine interleukin-12-treated mice, decreased brain lesions and parasite load were associated with antigen-specific antibody IgG2a > IgG1 and a three-fold increased ratio of antigen-specific interferon-gamma:interleukin-4 from splenocytes; the interleukin-12 effect was dependent upon interferon-gamma, as indicated by concomitant in vivo interferon-gamma neutralisation. By 6 weeks p.i. with N. caninum, there were no differences in brain lesions and parasite load between interleukin-12- and PBS-treated groups, indicating that the effects of interleukin-12 on driving a protective type 1 response were transient. These data indicate a role for interferon-gamma, interleukin-12 and type 1 immune responses in control of acute neosporosis in mice.     

Age-related changes of the susceptibility to infection with Brugia pahangi in male and female BALB/c mice

Effects of host age and sex on murine Brugia pahangi infection were examined. In female BALB/c mice, partial resistance to B. pahangi developed rapidly with age. In animals inoculated at 5-6 wk of age, resistance was about 2 times higher than in the youngest group (2-3 wk) and remained at a similar level through the study period (12 wk). Male BALB/c mice showed a similar tendency until 5-6 wk of age, but afterward their susceptibility increased with age. Significant difference in the susceptibility was detected between sexes after sexual maturation. After macrophage blockade by an injection of carbon particles into the mice, the age- and sex-related differences were completely abolished. This suggests that macrophages might have an important role(s) in the age- and sex-related changes of susceptibility to B. pahangi in BALB/c mice.     

Non-ulcerative cutaneous lesion in immunodeficient mice with Leishmania amazonensis infection

Cutaneous leishmaniasis begins as papules or nodules at the site of promastigote inoculation. The next key pathogenic event in this disease is the formation of an ulcer at this site. Leishmania infection in immunodeficient mice, however, showed non-ulcerative cutaneous lesions suggesting the involvement of the immune system in ulcer formation. Severe combined immunodeficient (SCID), recombination-activating gene 2 knockout (RAG-2-/-), and immunocompetent mice were inoculated subcutaneously with cultured L. amazonensis promastigotes. Macroscopic nodules appeared at the inoculation site within 2 weeks of infection in all the mice and gradually extended to the surrounding skin tissue. Although nodules of immunocompetent mice ulcerated within 6 weeks, immunodeficient mice did not form ulcers even after 25 weeks of inoculation. These results strongly suggest the importance of functional T and B cells in ulcer formation of cutaneous leishmaniasis and are consistent with clinical features of non-ulcerative cutaneous leishmaniasis in some AIDS patients. The present study also indicates that the L. amazonensis-infected immunodeficient mouse model might be suitable for studying the mechanisms of ulcer formation in cutaneous leishmaniasis.