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 2012, 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. Ex vivo analyses of neutrophils recovered from the L. major loaded dermis found that the uptake of L. major metacyclic promastigotes accelerated neutrophil apoptosis. Infected neutrophils were preferentially captured by dermal DCs when injected back into the mouse ear dermis, and stained positive for neutrophil markers 24 h following dermal challenge with L. major directly, indicating that the DCs 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. MerTk, a member of the family of receptor tyrosine kinases, was shown to be involved in the suppression of DC activation following their capture of infected neutrophils in vitro and 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. We previously reported that a whole cell, autoclaved killed plus CpG ODN vaccine that conferred protection against needle challenge with L. major failed to protect mice against infected sand fly challenge. In 2012, we extended these studies to include the only defined, sub-unit vaccine currently in clinical trial, and similarly found that while recombinant Leishmania poly-proteins delivered with the TLR 4 agonists monophosphoryl lipid A (MPL) or glucopyranosyl lipid A (GLA) protected mice against L. major delivered by needle, mice exposed to infected sand flies were not protected. Vaccinated mice had more severe lesions, associated with greater numbers of neutrophils and CD4+IL-17+ T cells at the challenge site. Only mice with healed primary infections were significantly protected against infected sand fly challenge. Pivotal to the generation of an effective vaccine against Leishmaniasis will be a more thorough understanding of the subsets and function of the cells that are generated by a healed primary infection that protect against natural sand fly challenge. Studies carried out in 2012 revealed that T cells displaying properties of Effector cells and not Central Memory cells distinguish protective from non-protective immune responses. During a protective response, antigen-specific cells migrated rapidly to the dermal site of challenge in an antigen-independent manner, they did not proliferate, and they were primarily CD44hi, CD62Llow, LY6C+, and IFNg single producers. Adoptive transfer of these cells to naive mice conferred protection. These observations suggest that rapidly recruited CD4+ T Eff cells already present in the recirculating pool at the time of challenge are critical to protection against sand fly transmitted infection, emphasizing the likely role of persisting antigen in maintaining protective T cell immunity, with direct implications for vaccine design. Experimental mouse models are also being used to understand the host responses that control non-healing and visceralizing forms of Leishmaniasis. Infection of C57BL/6 mice with most Leishmania major strains such as Lm Friedlin (Fn), results in a healing lesion with minimal pathology observed at the site of parasite inoculaation in the skin. However, following infection with the L.m. NIH/Sd strain from West Africa, C57BL/6 mice, despite their strongly polarized Th1 response and comparable growth of the parasite during the acute stage of infection, exhibit non-healing lesions and severe pathology leading to complete destruction of the ear dermis. In comparison to Lm Fn, the pathology due to Lm Sd is preceded by a greater recruitment of CD4+ and CD8+granzymeB+ T cells, and especially neutrophils to the site, and with upregulated expression of multiple cytokines, including IL-17, IL-27, IL-10, and IL-1a/b. Lm Sd infection of IL-17a-/- mice resulted in complete tissue destruction, whereas infection of IL-1Ra-/- mice resulted in minimal pathology. The L. major sub-strain specific molecules expressed by the parasite that drive the IL-1a/b dependent pathology are being mapped using the Lm Fn x Lm Sd hybrid clones that segregate these phenotypes. Hybrid progeny generated in sand flies co-infected with L. major and L. infantum are also being used to map the genes controlling tissue tropism. In C57BL/6, intradermal injection of 2.106 L. infantum results in dissemination of the parasite from the skin to the liver and spleen during 3 weeks post-infection, whereas L. major fails to disseminate. Employing this model, 3 of 6 hybrid clones were able to disseminate, while the other half remained confined to skin and draining lymph node, suggesting that a single or closely linked set of genes control visceral dissemination. Depressed cell mediated immunity in human visceral leishmaniasis (VL), revealed as the inability of peripheral blood mononuclear cells (PBMCs) to respond to Leishmania antigen, remains a hallmark of and is thought to underlie the progressive nature of this disease. We have recently reported the ability of a whole blood, IFN gamma release assay to detect sub-clinical infections amongst healthy individuals living in the kala-azar endemic zone in Bihar, India, and the surprising result that patients with active VL also secreted significant levels of antigen-specific IFNg in this assay. We were interested to extend these findings to a larger cohort of subjects, and to employ the whole blood assay to detect additional cytokines that might better correlate with the disease status of infected individuals. We evaluated IFNg, TNFa, and IL-10 release in 35 patients with active VL, 54 cured VL, 27 patients with other diseases, 52 Non-Endemic Healthy Controls, and 147 Endemic Healthy Controls. The whole blood cells from the majority of both active (80%) and cured (85%) VL patients, as well as 24% of EHCs with presumed sub-clinical infections, produced significantly elevated levels of IFNg. The findings do not support a severe Th1 response defect in kala-azar. Importantly, only the active VL patients also produced IL-10, which in conjunction with IFNg better reflects the immune responses that distinguish active cases from cured or sub-clinically infected, immune individuals.

Project Start
Project End
Budget Start
Budget End
Support Year
26
Fiscal Year
2012
Total Cost
$818,932
Indirect Cost
City
State
Country
Zip Code
Singh, Neetu; Kumar, Rajiv; Engwerda, Christian et al. (2016) Tumor necrosis factor alpha neutralization has no direct effect on parasite burden, but causes impaired IFN-γ production by spleen cells from human visceral leishmaniasis patients. Cytokine 85:184-90
Charmoy, Melanie; Hurrell, Benjamin P; Romano, Audrey et al. (2016) The Nlrp3 inflammasome, IL-1β, and neutrophil recruitment are required for susceptibility to a nonhealing strain of Leishmania major in C57BL/6 mice. Eur J Immunol 46:897-911
Faleiro, Rebecca J; Kumar, Rajiv; Bunn, Patrick T et al. (2016) Combined Immune Therapy for the Treatment of Visceral Leishmaniasis. PLoS Negl Trop Dis 10:e0004415
Ribeiro-Gomes, F L; Romano, A; Lee, S et al. (2015) Apoptotic cell clearance of Leishmania major-infected neutrophils by dendritic cells inhibits CD8⁺ T-cell priming in vitro by Mer tyrosine kinase-dependent signaling. Cell Death Dis 6:e2018
Sacks, David L; Melby, Peter C (2015) Animal models for the analysis of immune responses to leishmaniasis. Curr Protoc Immunol 108:19.2.1-19.2.24
Romano, Audrey; Doria, Nicole A; Mendez, Jonatan et al. (2015) Cutaneous Infection with Leishmania major Mediates Heterologous Protection against Visceral Infection with Leishmania infantum. J Immunol 195:3816-27
Sacks, David L (2014) Vaccines against tropical parasitic diseases: a persisting answer to a persisting problem. Nat Immunol 15:403-5
Peters, Nathan C; Pagán, Antonio J; Lawyer, Phillip G et al. (2014) Chronic parasitic infection maintains high frequencies of short-lived Ly6C+CD4+ effector T cells that are required for protection against re-infection. PLoS Pathog 10:e1004538
Ribeiro-Gomes, Flavia L; Roma, Eric Henrique; Carneiro, Matheus B H et al. (2014) Site-Dependent Recruitment of Inflammatory Cells Determines the Effective Dose of Leishmania major. Infect Immun 82:2713-27
Sacks, David (2014) Lost but not forgotten. Cell Host Microbe 16:423-5

Showing the most recent 10 out of 20 publications