Paola M. Boggiatto

Transplacental Transmission of Leishmania infantum as a Means for Continued Disease Incidence in North America

Background Dogs are the predominant domestic reservoir for human L. infantum infection. Zoonotic visceral leishmaniasis (ZVL) is an emerging problem in some U.S. dog breeds, with an annual quantitative PCR prevalence of greater than 20% within an at-risk Foxhound population. Although classically Leishmania is transmitted by infected sand flies and phlebotomine sand flies exist in the United States, means of ongoing L. infantum transmission in U.S. dogs is currently unknown. Possibilities include vertical (transplacental/transmammary) and horizontal/venereal transmission. Several reports have indicated that endemic ZVL may be transmitted vertically. Aims Our aims for this present study were to establish whether vertical/transplacental transmission was occurring in this population of Leishmania-infected US dogs and determine the effect that this means of transmission has on immune recognition of Leishmania. Methodology A pregnant L. infantum-infected dam donated to Iowa State University gave birth in-house to 12 pups. Eight pups humanely euthanized at the time of birth and four pups and the dam humanely euthanized three months post-partum were studied via L. infantum-kinetoplast specific quantitative PCR (kqPCR), gross and histopathological assessment and CD4+ T cell proliferation assay. Key Results This novel report describes disseminated L. infantum parasites as identified by kqPCR in 8 day old pups born to a naturally-infected, seropositive U.S. dog with no travel history. This is the first report of vertical transmission of L. infantum in naturally-infected dogs in North America, emphasizing that this novel means of transmission could possibly sustain infection within populations. Major Conclusions Evidence that vertical transmission of ZVL may be a driving force for ongoing disease in an otherwise non-endemic region has significant implications on current control strategies for ZVL, as at present parasite elimination efforts in endemic areas are largely focused on vector-borne transmission between canines and people. Determining frequency of vertical transmission and incorporating canine sterilization with vector control may have a more significant impact on ZVL transmission to people in endemic areas than current control efforts.

Immunologic Indicators of Clinical Progression during Canine Leishmania infantum Infection

ABSTRACT In both dogs and humans Leishmania infantum infection is more prevalent than disease, as infection often does not equate with clinical disease. Previous studies additively indicate that advanced clinical visceral leishmaniasis is characterized by increased production of anti-Leishmania antibodies, Leishmania-specific lymphoproliferative unresponsiveness, and decreased production of gamma interferon (IFN-γ) with a concomitant increase of interleukin-10 (IL-10). In order to differentiate infection versus progressive disease for better disease prognostication, we temporally evaluated humoral and cellular immunologic parameters of naturally infected dogs. The work presented here describes for the first time the temporal immune response to natural autochthonous L. infantum infection in foxhounds within the United States. Several key changes in immunological parameters should be considered when differentiating infection versus clinical disease, including a dramatic rise in IgG production, progressive increases in antigen-specific peripheral blood mononuclear cell proliferation, and IFN-γ production. Polysymptomatic disease is precluded by increased IL-10 production and consistent detection of parasite kinetoplast DNA in whole blood. This clinical presentation and the immuno-dysregulation mirror those observed in human patients, indicating that this animal model will be very useful for testing immunomodulatory anti-IL-10 and other therapies.

Altered dendritic cell phenotype in response to Leishmania amazonensis amastigote infection is mediated by MAP kinase, ERK.

Initiation of productive immune responses against Leishmania depends on the successful transition of dendritic cells (DC) from an immature to a mature phenotype. This process is characterized by high CD40 surface expression as well as interleukin-12 production, which are frequently seen in response to L. major infection. In vivo footpad infection of C3HeB/FeJ mice for 7 days with L. amazonensis promoted an immature CD11c(+) DC phenotype characterized by both significantly low CD40 surface expression and significantly decreased interleukin-12p40 production compared with L. major infection of these same mice. In vitro infection of bone marrow-derived dendritic cells with L. amazonensis amastigotes resulted in rapid and significant phosphorylation of the mitogen activated protein kinase, extracellular signal-regulated kinase 1/2, observed within minutes of exposure to the parasite. Infection with L. amazonensis promastigotes led to increased 1/2 phosphorylation after 4 hours of infection compared with L. major infection, which correlated with promastigote transformation into amastigotes. Treatment of bone marrow-derived dendritic cells with a mitogen activated protein kinase kinase-specific inhibitor, PD98059, led to regained surface CD40 expression and interleukin-12p40 production following L. amazonensis amastigote infection compared with non-treated, infected DC. Treatment of L. amazonensis-infected mice with the highly-specific mitogen activated protein kinase kinase inhibitor, CI-1040, enhanced surface CD40 expression on CD11c(+) DC obtained from the draining lymph node. L. amazonensis amastigotes, through activation of extracellular signal-regulated kinase 1/2, inhibit the ability of DC to undergo proper maturation both in vitro and in vivo.

Pathogen-Derived Oligosaccharides Improve Innate Immune Response to Intracellular Parasite Infection

Pathogen glycolipids, including Leishmania spp. lipophosphoglycan (LPG) and Mycobacterium tuberculosis mannosylated lipoarabinomannan (ManLAM), modulate essential interactions with host phagocytic cells. Polysaccharide and lipid components promote immunomodulation. Owing to the stereochemistry required to synthesize oligosaccharides, the roles for oligosaccharides in the pathogenesis of infectious diseases have remained largely unknown. Recent advances in carbohydrate chemistry allowed us to synthesize pathogen surface oligosaccharides to discern their immune response-altering activities. Trimannose cap carbohydrates from ManLAM and LPG altered the production of proinflammatory cytokines via a toll-like receptor (TLR2)-mediated mechanism in vitro and in vivo. In vivo treatment with trimannose led to increased Th1-polarizing, IL-12p40-producing cells from the draining lymph nodes of treated Leishmania major-infected mice compared with cells from untreated infected mice. Trimannose treatment increased the production of other Th1 proinflammatory cytokines (ie, interferon-γ, IL-6, and tumor necrosis factor-α) critical for a productive immune response to either pathogen. This significant difference in cytokine production between trimannose cap sugar-treated and control groups was not observed in draining lymph node cells from TLR2(-/-) mice. Type of inflammation and rate of bead entry into macrophages and dendritic cells were different for trimannose-coated beads compared with control oligosaccharide-coated beads, indicating selective lectin receptor/oligosaccharide interactions mediating cell entry and cytokine production. These novel findings may prompt the development of targeted oligosaccharide adjuvants against chronic infections.

Safety and Biocompatibility of Carbohydrate-Functionalized Polyanhydride Nanoparticles

ABSTRACTCarbohydrate functionalization of nanoparticles allows for targeting of C-type lectin receptors. This family of pattern recognition receptors expressed on innate immune cells, such as macrophages and dendritic cells, can be used to modulate immune responses. In this work, the in vivo safety profile of carbohydrate-functionalized polyanhydride nanoparticles was analyzed following parenteral and intranasal administration in mice. Polyanhydride nanoparticles based on 1,6-bis-(p-carboxyphenoxy)hexane and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane were used. Nanoparticle functionalization with di-mannose (specifically carboxymethyl-α-d-mannopyranosyl-(1,2)-d-mannopyranoside), galactose (specifically carboxymethyl-β-galactoside), or glycolic acid induced no adverse effects after administration based on histopathological evaluation of liver, kidneys, and lungs. Regardless of the polymer formulation, there was no evidence of hepatic or renal damage or dysfunction observed in serum or urine samples. The histological profile of cellular infiltration and the cellular distribution and kinetics in the lungs of mice administered with nanoparticle treatments followed similar behavior as that observed in the lungs of animals administered with saline. Cytokine and chemokine profiles in bronchoalveolar lavage fluid indicated surface chemistry dependence on modest secretion of IL-6, IP-10, and MCP-1; however, there was no evidence of any deleterious histopathological changes. Based on these analyses, carbohydrate-functionalized nanoparticles are safe for in vivo applications. These results provide foundational information towards the evaluation of the capabilities of these surface-modified nanoparticles as vaccine delivery formulations.

An In Vitro Model of Antibody-Enhanced Killing of the Intracellular Parasite Leishmania amazonensis

Footpad infection of C3HeB/FeJ mice with Leishmania amazonensis leads to chronic lesions accompanied by large parasite loads. Co-infecting these animals with L. major leads to induction of an effective Th1 immune response that can resolve these lesions. This cross-protection can be recapitulated in vitro by using immune cells from L. major-infected animals to effectively activate L. amazonensis-infected macrophages to kill the parasite. We have shown previously that the B cell population and their IgG2a antibodies are required for effective cross-protection. Here we demonstrate that, in contrast to L. major, killing L. amazonensis parasites is dependent upon FcRγ common-chain and NADPH oxidase-generated superoxide from infected macrophages. Superoxide production coincided with killing of L. amazonensis at five days post-activation, suggesting that opsonization of the parasites was not a likely mechanism of the antibody response. Therefore we tested the hypothesis that non-specific immune complexes could provide a mechanism of FcRγ common-chain/NADPH oxidase dependent parasite killing. Macrophage activation in response to soluble IgG2a immune complexes, IFN-γ and parasite antigen was effective in significantly reducing the percentage of macrophages infected with L. amazonensis. These results define a host protection mechanism effective during Leishmania infection and demonstrate for the first time a novel means by which IgG antibodies can enhance killing of an intracellular pathogen.

Promotion of a Functional B Cell Germinal Center Response after Leishmania Species Co-Infection Is Associated with Lesion Resolution

Co-infection of C3HeB/FeJ (C3H) mice with both Leishmania major and Leishmania amazonensis leads to a healed footpad lesion, whereas co-infection of C57BL/6 (B6) mice leads to non-healing lesions. This inability to heal corresponds to a deficiency in B cell stimulation of the macrophage-mediated killing of L. amazonensis in vitro and a less robust antibody response. The mechanism that leads to healing of these lesions is not completely known, although our studies implicate the B cell response as having an important effector function in killing L. amazonensis. To understand more completely this disparate clinical outcome to the same infection, we analyzed the draining lymph node germinal center B cell response between co-infected C3H and B6 mice. There were more germinal center B cells, more antibody isotype-switched germinal center B cells, more memory B cells, and more antigen-specific antibody-producing cells in co-infected C3H mice compared to B6 mice as early as 2 weeks postinfection. Interleukin (IL)-21 production and IL-21 receptor expression in both mouse strains, however, were similar at 2 weeks, suggesting that the difference in the anti-Leishmania response in these mouse strains may be due to differences in T follicular cell commitment or intrinsic B cell differences. These data support the idea that functional B cells are important for healing L. amazonensis in this infectious disease model.

Lung Deposition and Cellular Uptake Behavior of Pathogen‐Mimicking Nanovaccines in the First 48 Hours

Pulmonary immunization poses the unique challenge of balancing vaccine efficacy with minimizing inflammation in the respiratory tract. While previous studies have shown that mice immunized intranasally with F1-V-loaded polyanhydride nanoparticles are protected from a lethal challenge with Yersinia pestis, little is known about the initial interaction between the nanoparticles and immune cells following intranasal administration. Here, the deposition within the lung and internalization by phagocytic cells of polyanhydride nanovaccines encapsulating F1-V are compared with that of soluble F1-V alone or F1-V adjuvanted with monophosphoryl lipid A (MPLA). Encapsulation of F1-V into polyanhydride nanoparticles prolonged its presence while F1-V administered with MPLA is undetectable within 48 h. The inflammation induced by the polyanhydride nanovaccine is mild compared with the marked inflammation induced by the MPLA-adjuvanted F1-V. Even though F1-V delivered with saline is detected in the lung 48 h after administration, it is known that this regimen does not elicit a protective immune response. The prolonged F1-V presence in the lung in concert with the mild inflammatory response provided by the nanovaccine provides new insights into the development of protective immune responses with a single intranasal dose.

A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis

Infection of C3HeB/FeJ and C57BL/6 mice with Leishmania major stimulates a healing cell-mediated immune response, while Leishmania amazonensis infection leads to chronic disease. Here we show C3HeB/FeJ mice co-infected with both species of Leishmania heal, while co-infected C57BL/6 mice do not. Using an in vitro killing assay we determined B cells from infected C57BL/6 mice are ineffective in promoting parasite killing compared with B cells from infected C3HeB/FeJ mice. Furthermore, infected C57BL/6 mice produce less antigen-specific antibodies compared with infected C3HeB/FeJ mice. These findings suggest B cells play a required role in the cell-mediated immune response against L. amazonensis.

Targeted extracellular signal-regulated kinase activation mediated by Leishmania amazonensis requires MP1 scaffold

Leishmania amazonensis infection promotes alteration of host cellular signaling and intracellular parasite survival, but specific mechanisms are poorly understood. We previously demonstrated that L. amazonensis infection of dendritic cells (DC) activated extracellular signal-regulated kinase (ERK), an MAP-kinase kinase kinase, leading to altered DC maturation and non-healing cutaneous leishmaniasis. Studies using growth factors and cell lines have shown that targeted, robust, intracellular phosphorylation of ERK1/2 from phagolysosomes required recruitment and association with scaffolding proteins, including p14/MP1 and MORG1, on the surface of late endosomes. Based on the intracellular localization of L. amazonensis within a parasitophorous vacuole with late endosome characteristics, we speculated that scaffolding proteins would be important for intracellular parasite-mediated ERK signaling. Our findings demonstrate that MP1, MORG1, and ERK all co-localized on the surface of parasite-containing LAMP2-positive phagolysosomes. Infection of MEK1 mutant fibroblasts unable to bind MP1 demonstrated dramatically reduced ERK1/2 phosphorylation following L. amazonensis infection but not following positive control EGF treatment. This novel mechanism for localization of intracellular L. amazonensis-mediated ERK1/2 phosphorylation required the endosomal scaffold protein MP1 and localized to L. amazonensis parasitophorous vacuoles. Understanding how L. amazonensis parasites hijack host cell scaffold proteins to modulate signaling cascades provides targets for antiprotozoal drug development.