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BALB/c mice were given or not iron around the time of intradermal parasite inoculation, in their ears, of either 10(6) stationary-phase (designated "high-dose model") or 10(3)Leishmania major metacyclic promastigotes (designated "low-dose model"). Iron-loaded mice in the high-dose model displayed delayed and limited pathogenic processes, whereas in the low-dose model, the mice remained ear lesion-free over 12 months post-parasite inoculation. These phenotypes were coupled to an increased leukocyte oxidative burst displayed mainly by neutrophils: it was early and transient in the high-dose model, whereas it was sustained in the low-dose model. In the latter model, injection of an antioxidant (diphenyleneiodonium chloride) at week 2 post-L. major inoculation resulted in a significant decrease in oxidative burst and reversed the protective status. The increased and sustained oxidative burst displayed by the neutrophils, the sustained presence of IL-12 (p40/p70)-positive leukocytes in the ear dermis, the low number of inflammatory leukocytes in the ear dermis and their concomitant high number in the draining lymph node are three related features that likely contribute to the shaping of the protective status, the onset and dynamic maintenance of which are antioxidant sensitive.

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.

Systemic iron delivery to BALB/c mice, at time points surrounding the inoculation of 1000 Leishmania major metacyclic promastigotes intradermally in the ear results in the complete absence of onset and further development of ear lesion. In these iron-protected mice, the L. major intracellular progeny remains very low in both the ear and the draining lymph node. The iron-induced protective status is associated with a diphenyleneiodonium-sensitive sustained increased oxidative burst. We showed that iron-loaded mice developed no lesions at the site of the primary inoculation and were also resistant to reinoculation at a distant site (intradermal re-inoculation of 1000 metacyclic promastigotes in the contra-lateral ear). Interestingly, in the lymph node cell population recovered from iron-loaded mice at weeks 8 and 12 after the second parasite inoculation, and whatever the protective status studied--primary or resistant to re-inoculation--three potentially related features were observed: (i) NF-kappaB activation, (ii) enhanced TCR-mediated T lymphocyte proliferation, and (iii) high number of IFN-gamma-positive CD4(+)T cells. These results show a putative role of an iron-induced reactive oxygen species-dependent activation of NF-kappaB in the development of protective immunity against L. major.

We previously established that Leishmania promastigotes express a transferrin receptor and that iron chelators inhibit promastigote growth in vitro. Thus, we were interested in modulating the vertebrate host iron pool and to monitor whether such changes will affect the outcome of L. major infection in BALB / c mice, inoculated in the footpad with 106 stationary phase promastigotes. Treatment of mice with desferrioxamine resulted in a slight delay of the development of cutaneous lesions. In contrast and unexpectedly, systemic iron delivery, at early time points of parasite delivery, significantly limited footpad pathology. Accordingly, parasite loads at the site of parasite delivery, the draining lymph node, liver and spleen were significantly reduced in iron-loaded mice. Importantly, the "protective" effect of iron delivery correlated with the presence, at the site of inoculation, of lower levels of IL-4 and IL-10 transcripts while both IFN-gamma and inducible nitric oxide synthase transcripts were at higher levels. The presence of more type 1 cytokine transcripts was further supported by the increased levels of IgG2a in their sera. These data strongly suggest that susceptibility to L. major as assessed in the footpad model is modifiable by interventions that alter the iron status of the host at early time points of parasite delivery.