Our studies have emphasized the phenotype, functionality and life-span of the effector and memory T cells from mice with healed primary infections that are able to transfer immunity. Studies in the past year have revealed a role for neutrophils and skin resident memory T cells (TRM) in the rapid expression of immunity in the skin of healed mice. Since antigen-primed tissue resident memory T cells are already existent within skin of previously infected mice and an abundance of neutrophils migrate to the infection site within minutes of needle or sand fly challenge, we hypothesized an in vivo role of infected neutrophils as antigen presenting cells for rapid activation of TRM during secondary infections. Our results show that within 4 hours of secondary infection, skin TRM cells express the activation marker CD25 in a neutrophil-dependent fashion. We could also show that at this early time after challenge neutrophils express low but detectable levels of MHC II and co-stimulatory molecules. Our data point to a previously unknown collaborative function of neutrophils in activation of tissue resident T cells whose main function is an accelerated protection against secondary infectious challenge. The Th2 dominant response that develops in BALB/c mice infected with L. major is the classical experimental model to study the immune dysfunction underlying uncontrolled Leishmania infections. However, this experimental model has failed to adequately explain chronic and non-healing forms of leishmanisis in humans, which are associated with strong expression of pro-inflammatory cytokines, such IFN gamma and TNF alpha. We have developed a model of non-healing cutaneous leishmaniasis in conventionally resistant C57BL/6 mice infected with L. major Seidman strain (LmSd) that was isolated from a patient with non-healing cutaneous lesions. Our recent studies have revealed a role for Nlrp3 inflammasome dependent IL-1 beta in the evolution of the non-healing response. We have also identified a population of embryonic-derived M2-like dermal-resident macrophages that are present under steady state conditions and that are preferentially infected by the LmSd strain. The favored infection of M2-like dermal and bone marrow derived macrophages by LmSd was mannose dependent, and genetic deletion of the mannose receptor (MR) reversed the non-healing phenotype produced by LmSd in C57Bl/6 mice. The MR high dermis-resident macrophages are maintained and expanded locally by IL-1 beta induced IL-4 and IL-10 during infection. The studies are the first to show that Leishmania exploit M2-like resident macrophages in the skin to evade anti-microbial immunity and produce chronic cutaneous disease. As most currently licensed vaccines work by eliciting humoral responses that can be maintained for long periods even in the absence of persisting antigen, an anti-Leishmania vaccine that can rely on antibodies to inhibit the infectious process in the mammalian host might overcome one of the main barriers to the development of an effective T cell based vaccine. We have explored the possibility that antibodies targeting the infective, metacyclic promastigotes inoculated by the sand fly vector might be inhibitory to the earliest stages of infection in the mammalian host. We have data to show that passive immunization of mice with a monoclonal antibody directed against the common phosphoglycan chains of lipophosphoglycan (LPG) and proteophosphoglycan (PPG), representing the major surface and secreted molecules of Leishmania promastigotes, conferred complete protection against cutaneous leishmaniasis due to Leishmania major transmitted by infected sand flies. Current studies are designed to extend this vaccination strategy to the visceral form of disease in hamsters; specifically, to demonstrate the ability of a B-cell based vaccine targeting the LPG/PPG expressed by the infectious stage of L. donovani to protect hamsters against VL transmitted by infected sand flies. Current drugs against visceral leishmaniasis (VL) are costly and are associated with severe side effects. Modulation of immune responses to facilitate cure of leishmaniasis has been suggested as a strategy to improve VL therapy. However, the immune events mediating progression or control of Leishmania donovani during VL remain unclear in humans. Elevated co-expression of TNF alpha and IL-10 has been a consistent finding in VL, and while the up-regulation of IL-10 is important to limit tissue pathology, it also can facilitate their replication in macrophages. We have shown that blocking IL-10 promotes killing of L. donovani in short term cultures of splenic aspirate cells from patients with active disease. We have also shown that blocking IFN gamma prevents parasite killing in these cultures. The protective role of TNF-a in human VL is not clear, and contrary to expectations, we found no direct effect of TNF alpha neutralization on parasite burden in ex vivo splenic aspirate cultures, arguing that it may not be a suitable target for host direct therapy in VL.

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Lee, Sang Hun; Charmoy, Melanie; Romano, Audrey et al. (2018) Mannose receptor high, M2 dermal macrophages mediate nonhealing Leishmania major infection in a Th1 immune environment. J Exp Med 215:357-375
Romano, Audrey; Carneiro, Matheus B H; Doria, Nicole A et al. (2017) Divergent roles for Ly6C+CCR2+CX3CR1+ inflammatory monocytes during primary or secondary infection of the skin with the intra-phagosomal pathogen Leishmania major. PLoS Pathog 13:e1006479
Charmoy, Melanie; Hurrell, Benjamin P; Romano, Audrey et al. (2016) The Nlrp3 inflammasome, IL-1?, and neutrophil recruitment are required for susceptibility to a nonhealing strain of Leishmania major in C57BL/6 mice. Eur J Immunol 46:897-911
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-? production by spleen cells from human visceral leishmaniasis patients. Cytokine 85:184-90
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
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
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
Singh, Om Prakash; Hasker, Epco; Sacks, David et al. (2014) Asymptomatic Leishmania infection: a new challenge for Leishmania control. Clin Infect Dis 58:1424-9
Gautam, Shalini; Kumar, Rajiv; Singh, Neetu et al. (2014) CD8 T cell exhaustion in human visceral leishmaniasis. J Infect Dis 209:290-9

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