Inoculation of Leishmania major by the bite of a natural sand fly vector, or by needle injection, induces an intense neutrophilic infiltrate into the skin. Neutrophils subsequently phagocytose the majority of deposited parasites but fail to kill them, a process that promotes sand fly transmitted infection. Neutrophil infiltration occurs whether parasites are present or not, suggesting the predominance of L.m.-infected neutrophils is a consequence of the host response to tissue injury. We employed mice deficient in critical components of several inflammatory and immunologic processes in an effort to indentify factors involved in neutrophil recruitment and parasite phagocytosis. Surprisingly, CXCR2 expression on neutrophils, an important chemokine receptor for neutrophil function in other experimental systems, did not play a direct role in the early recruitment and phagocytic capacity of neutrophils following co-transfer of CXCR2-/- and WT-control neutrophils into WT mice. Normal neutrophil recruitment and parasite uptake was also observed in MyD88 KO mice, and exposure of neutrophils to LPS in-vitro did not enhance parasite phagocytosis. In contrast, mast cell deficient mice showed impaired neutrophil recruitment, while C3 deficient mice showed impaired parasite uptake. In-vitro, phagocytosis of Leishmania by neutrophils was highly C3 dependent. Our findings begin to address the critical components of the neutrophil response to Leishmania deposition in the skin. Since neutrophils are the predominant recruited and infected cells in the early stages of Leishmania major infection in the skin, we were interested to study the possible interaction of neutrophils and dendritic cells (DC) in vivo and the consequences of this interaction for the immune response against Leishmania. Following i.d. inoculation of RFP-expressing L. major into lysozyme-GFP mice, the recovered GFPhi neutrophils in the dermal infiltrate were sorted into uninfected and infected populations, and reinjected in the ear dermis of C57BL/6 mice. Amongst the dermal RFP+ cells recovered 4 hrs later, only the DCs were GFP+, suggesting preferential uptake of parasitized neutrophils by DCs in vivo. Moreover, after direct injection of RFP-L. major, the RFP+DCs recovered from the skin had also acquired neutrophil markers, myeloperoxidase and elastase. By contrast, RFP+DCs recovered from mice treated with a specific neutrophil depleting Ab (1A8), remained negative for these markers. These data indicate that the normal early encounter of DC with L. major in the skin is in the context of infected neutrophils. Neutrophil depletion 1d prior to infection with OVA expressing L. major strongly enhanced priming of adoptively transferred OT-II CD4+ T cells in vivo, suggesting down modulation of APC function following uptake of parasitized neutrophils in vivo. These data demonstrate a previously unrecognized role of neutrophils in immune evasion. Numerous experimental vaccines have been developed to protect against infection with Leishmania, but a human vaccine still does not exist. Only rarely has the efficacy of anti-Leishmania vaccines been properly evaluated under experimental conditions following natural vector-transmission by infected sand fly bite. We observed that in C57Bl/6 mice with healed L. major infections, parasite-specific, cytokine-producing CD4+ T cells were found at the site of challenge by infected sand fly bite within 24 hours, and these mice were highly resistant to sand fly transmitted infection. In contrast, mice vaccinated with autoclaved L. major antigen (ALM)+CpG oligodeoxynucleotides or a recombinant protein vaccine, Leish-111f in MPL-SE that is currently in clinical trials and that protected against needle inoculation of parasites, showed delayed recruitment of protective cells and completely failed to protect against infected sand fly challenge. In addition, the protection conferred against needle challenge by the non-living vaccines was in no instance as strong as the resistance to secondary challenge displayed by the healed mice. Sand fly, but not needle, challenge, resulted in the maintenance of a localized neutrophilic response at the inoculation site, and removal of neutrophils following vector transmission led to increased parasite-specific immune responses and promoted the efficacy of the killed vaccine. These observations begin to identify the critical factors influencing vaccine efficacy following natural transmission of Leishmania, and establish the acquired resistance resulting from healed infections as the gold standard for potency that experimental vaccines may need to achieve. We have previously addressed the cellular source of elevated IL-10 in human visceral leishmaniasis (VL), and identified CD4+CD25-Foxp3- T cells as the major producers of IL-10 in the VL spleen. The findings suggest that Tr1 cells, or Th subsets that co-express IL-10, are important in suppression of anti-leishmanial immunity in human VL. In the past year, we have begun to study the factors regulating T cell IL-10 production in human VL. In the mouse, an IL-12 family member, IL-27, has been shown to promote IL-10 secretion by CD4+ T cells in vitro and in various inflammatory settings in vivo. We were able to detect elevated circulating levels of IL-27 in VL patients (fig. 1), as well as significantly elevated mRNA levels for both IL-27p28 and EBI-3 subunits in the VL spleen during active disease. Expression of IL-21 mRNA was also significantly enhanced in pre- as compared to post treatment splenic aspirate cells, and especially in comparison to cells from healthy spleens. The IL-27 transcripts were enriched in macrophages, while the IL-21 and IL-10 transcripts were enriched in T cells. The role for IL-21 in amplifying IL-27 induced, T cells IL-10 production has been recently recognized in mice. Furthermore, the ability of peripheral blood cells from VL patients to produce antigen-specific IL-10 was significantly enhanced in the presence of exogenous IL-21. These data are consistent with the possibility that IL-27 from innate cells induces IL-21 that acts as an autocrine growth factor to expand antigen-specific, IL-10 producing T cells in VL patients.
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