We previously reported that neutrophils are the predominant recruited and infected cells during in the early stages of Leishmania major infection in the skin, and that neutrophil depletion promotes host resistance to sand fly transmitted infection. In 2011, we carried out further experiments to address how the massive influx of neutrophils aimed at wound repair and sterilization, might modulate the function of dermal DCs and their ability to initiate anti-leishmanial immunity. The infected neutrophils recovered from the skin expressed elevated apoptotic markers compared to uninfected neutrophils, and were preferentially captured by dermal DCs when injected back into the mouse ear dermis. Following challenge with L. major directly, the majority of the infected DCs recovered from the skin at 24 hr stained positive for neutrophil markers, indicating that they acquired their parasites via uptake of infected neutrophils. When infected, dermal DCs were recovered from neutrophil depleted mice, their expression of activation markers was markedly enhanced, as was their capacity to present Leishmania antigens ex vivo. Neutrophil depletion also enhanced the priming of L. major specific CD4+ T cells in vivo. The findings suggest that following their rapid uptake by neutrophils in the skin, L. major exploits the immunosuppressive effects associated with the apoptotic cell clearance function of DCs to inhibit the development of acquired resistance until the acute neutrophilic response is resolved. Pivotal to the generation of a vaccine that protects against infection with Leishmania, is identification of cells that protect against natural transmission of the parasite by the insect vector, the sand fly. Sub-clinical chronic infection with Leishmania major is thought to generate two populations of CD4+ T cells that can contribute to protective secondary immunity following needle challeng: central memory (Cm) cells that do not require parasite persistence to be maintained, and effector (Eff) cells, that do. Studies carried out in 2011 revealed that T cells displaying properties of Eff and not Cm cells distinguish protective from non-protective immune responses following natural challenge by infected sand fly bite. During a protective response, antigen-specific cells migrated to a dermal site of challenge in an antigen-independent manner, did not require antigen re-stimulation to produce cytokine when antigen was present at the site, and could be found in the dermis within 24 hrs. Two-photon intra-vital microscopy and flow cytometric analysis revealed that, following adoptive transfer of sorted Cm and Eff CD4+ T cells obtained from mice with healed primary infections, Eff, but not Cm, cells were directly recruited to dermal sites of low dose parasite deposition. In contrast, Cm, but not Eff, cells accumulated in DLNs in large numbers. High proportions of adoptively transferred Eff cells recovered from the dermis co-produced IFN-gamma, TNF-alpha, and IL-2 in response to antigen re-stimulation. Although adoptively transferred Eff cells contained a high frequency of antigen-specific cells, their biggest impact appeared to be on the enhanced activation of antigen-specific nave cells. These observations suggest CD4+ T Eff cells are critical to mediating immunity to sand fly transmitted Leishmaniasis, emphasizing the role of persisting antigen in maintaining protective T cell immunity, with direct implications for vaccine design. The mechanisms underlying the failure to contain the growth of Leishmania parasites in human visceral leishmaniasis (VL) are not understood. We have previously described the elevated levels of the immunosuppressive cytokine, IL-10, in target organs and peripheral blood of patients with active VL in India, and identified CD4+CD25-Foxp3- T cells as the major producers of IL-10 in the VL spleen. In humans, elevated levels of IL-10 are correlated with other important chronic infectious diseases, including malaria, tuberculosis, and HIV. The evidence that IL-10 contributes to the chronicity of infection in humans is nonetheless indirect, and confined to the enhancement of immune correlates of protection when IL-10 function is blocked in vitro. There is so far no direct evidence in any clinical setting that IL-10 inhibition will promote pathogen clearance. In the past year, we have used the number of viable amastigotes present in splenic aspirate cells from VL patients, obtained routinely for diagnostic purposes, as a readout to explore the effect of IL-10 neutralization on parasite killing ex vivo. Following 3 days incubation of the splenic cells, the number of viable organisms in the anti-IL-10 treated cultures showed a highly significant reduction compared to the control treated cultures (control IgG, geometric mean 14540;anti-IL-10, geometric mean 1182;p <0.0001. Of the 67 paired cultures analyzed, 49 (73%) had fewer parasites, while 12 (18%) had more and 6 (9%) did not change under IL-10 neutralizing conditions. Furthermore, the IL-10 inhibition resulted in complete clearance of organisms from 20 (30%) of the cultures. Determination of the concentration of cytokines released by the spleen cells during the 3 days of culture revealed consistently elevated levels of both IFN gamma and TNF alpha in the anti-IL-10 treated cultures. These findings provide the most direct support to date for IL-10 as a therapeutic target in human VL. Visceral leishmaniasis has emerged as a public health concern in Tbilisi, the capital of Georgia. Over the past three years, seroepidemiological surveys were conducted to determine the prevalence and incidence of infection in children and dogs within the main focus of VL, and to identify risk factors associated with human infection. Of 4,250 children investigated, 7.3% were positive by direct agglutination test in a baseline survey;an apparent incidence rate of 6.0% was estimated by one year follow-up. None of the seropositive children progressed to VL during the survey. Increased seropositivity at one year was predicted by presence at baseline of clustered flying insects (OR=1.49;P=0.001), stray dogs (OR=1.33;P=0.023), and by persistent fever during the 6 months prior to baseline survey (OR=14.2;P<0.001). Overall, 18.2% (107/588) of domestic and 15.3% (110/718) of stray dogs were seropositive by the rk39 dipstick test. Parasites isolated from human and dog samples were identified by PCR analysis as Leishmania infantum. We conclude that there is a hyperactive focus of L. infantum transmission in Tbilisi with a high prevalence of human and canine infections.
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