Jessica A. Hess

Role of Eosinophils and Neutrophils in Innate and Adaptive Protective Immunity to Larval Strongyloides stercoralis in Mice

ABSTRACT The goal of this study was to determine the roles of eosinophils and neutrophils in innate and adaptive protective immunity to larval Strongyloides stercoralis in mice. The experimental approach used was to treat mice with an anti-CCR3 monoclonal antibody to eliminate eosinophils or to use CXCR2−/− mice, which have a severe neutrophil recruitment defect, and then determine the effect of the reduction or elimination of the particular cell type on larval killing. It was determined that eosinophils killed the S. stercoralis larvae in naïve mice, whereas these cells were not required for the accelerated killing of larvae in immunized mice. Experiments using CXCR2−/− mice demonstrated that the reduction in recruitment of neutrophils resulted in significantly reduced innate and adaptive protective immunity. Protective antibody developed in the immunized CXCR2−/− mice, thereby demonstrating that neutrophils were not required for the induction of the adaptive protective immune response. Moreover, transfer of neutrophil-enriched cell populations recovered from either wild-type or CXCR2−/− mice into diffusion chambers containing larvae demonstrated that larval killing occurred with both cell populations when the diffusion chambers were implanted in immunized wild-type mice. Thus, the defect in the CXCR2−/− mice was a defect in the recruitment of the neutrophils and not a defect in the ability of these cells to kill larvae. This study therefore demonstrated that both eosinophils and neutrophils are required in the protective innate immune response, whereas only neutrophils are necessary for the protective adaptive immune response to larval S. stercoralis in mice.

Eosinophils Act as Antigen-Presenting Cells to Induce Immunity to Strongyloides stercoralis in Mice

The objective of the present study was to explore the ability of eosinophils to present Strongyloides stercoralis antigen in naive and immunized mice. Antigen-pulsed eosinophils were injected intraperitoneally into naive or immunized mice, and then mice were examined for antigen-specific immune responses. A single inoculation of antigen-pulsed eosinophils was sufficient to prime naive mice and to boost immunized mice for antigen-specific T helper cell type 2 (Th2) immune responses with increased interleukin (IL)-4 and IL-5 production. Mice inoculated 3 times with live eosinophils pulsed with antigen showed significant increases in parasite antigen-specific immunoglobulin (Ig) M and IgG levels in their serum. Antigen-pulsed eosinophils deficient in major histocompatibility complex class II molecules or antigen-pulsed dead eosinophils failed to induce immune responses, thereby demonstrating the requirement for direct interaction between eosinophils and T cells. These experiments demonstrate that eosinophils function as antigen-presenting cells for the induction of the primary and the expansion of the secondary Th2 immune responses to S. stercoralis in mice.

Major basic protein from eosinophils and myeloperoxidase from neutrophils are required for protective immunity to Strongyloides stercoralis in mice.

ABSTRACT Eosinophils and neutrophils contribute to larval killing during the primary immune response, and neutrophils are effector cells in the secondary response to Strongyloides stercoralis in mice. The objective of this study was to determine the molecular mechanisms used by eosinophils and neutrophils to control infections with S. stercoralis. Using mice deficient in the eosinophil granule products major basic protein (MBP) and eosinophil peroxidase (EPO), it was determined that eosinophils kill the larvae through an MBP-dependent mechanism in the primary immune response if other effector cells are absent. Infecting PHIL mice, which are eosinophil deficient, with S. stercoralis resulted in development of primary and secondary immune responses that were similar to those of wild-type mice, suggesting that eosinophils are not an absolute requirement for larval killing or development of secondary immunity. Treating PHIL mice with a neutrophil-depleting antibody resulted in a significant impairment in larval killing. Naïve and immunized mice with neutrophils deficient in myeloperoxidase (MPO) infected with S. stercoralis had significantly decreased larval killing. It was concluded that there is redundancy in the primary immune response, with eosinophils killing the larvae through an MBP-dependent mechanism and neutrophils killing the worms through an MPO-dependent mechanism. Eosinophils are not required for the development or function of secondary immunity, but MPO from neutrophils is required for protective secondary immunity.

Complement Component C3 Is Required for Protective Innate and Adaptive Immunity to Larval Strongyloides stercoralis in Mice

This study examines the role of complement components C3 and C5 in innate and adaptive protective immunity to larval Strongyloides stercoralis in mice. Larval survival in naive C3−/− mice was increased as compared with survival in wild-type mice, whereas C3aR−/− and wild-type mice had equivalent levels of larval killing. Larval killing in naive mice was shown to be a coordinated effort between effector cells and C3. There was no difference between survival in wild-type and naive C5−/− mice, indicating that C5 was not required during the innate immune response. Naive B cell-deficient and wild-type mice killed larvae at comparable levels, suggesting that activation of the classical complement pathway was not required for innate immunity. Adaptive immunity was equivalent in wild-type and C5−/− mice; thus, C5 was also not required during the adaptive immune response. Larval killing was completely ablated in immunized C3−/− mice, even though the protective parasite-specific IgM response developed and effector cells were recruited. Protective immunity was restored to immunized C3−/− mice by transferring untreated naive serum, but not C3-depleted heat-inactivated serum to the location of the parasites. Finally, immunized C3aR−/− mice killed larvae during the adaptive immune response as efficiently as wild-type mice. Therefore, C3 was not required for the development of adaptive immunity, but was required for the larval killing process during both protective innate and adaptive immune responses in mice against larval S. stercoralis.

Human and Mouse Macrophages Collaborate with Neutrophils To Kill Larval Strongyloides stercoralis

ABSTRACT Macrophages are multifunctional cells that are active in TH1- and TH2-mediated responses. In this study, we demonstrate that human and mouse macrophages collaborate with neutrophils and complement to kill the parasite Strongyloides stercoralis in vitro. Infection of mice with worms resulted in the induction of alternatively activated macrophages (AAMϕ) within the peritoneal cavity. These cells killed the worms in vivo and collaborated with neutrophils and complement during the in vitro killing process. AAMϕ generated in vitro killed larvae more rapidly than naive macrophages, which killed larvae after a longer time period. In contrast, classically activated macrophages were unable to kill larvae either in vitro or in vivo. This study adds macrophages to the armamentarium of immune components that function in elimination of parasitic helminths and demonstrate a novel function by which AAMϕ control large extracellular parasites.

Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis

Neutrophils are multifaceted cells that are often the immune systems first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system.

Innate and adaptive immunity to the nematode Strongyloides stercoralis in a mouse model.

Mice have been used to the study the mechanisms of protective innate and adaptive immunity to larval Strongyloides stercoralis. During primary infection, neutrophils and eosinophils are attracted by parasite components and kill the larvae by release of granule products. Eosinophils also function as antigen-presenting cells for the induction of a Th2 response. B cells produce both IgM and IgG that collaborate with neutrophils to kill worms in the adaptive immune response. Vaccine studies have identified a recombinant diagnostic antigen that induced high levels of immunity to infection with S. stercoralis in mice. These studies demonstrate that there are redundancies in the mechanisms used by the immune response to kill the parasite and that a vaccine with a single antigen may be suitable as a prophylactic vaccine to prevent human strongyloidiasis.

Vaccines to combat river blindness: expression, selection and formulation of vaccines against infection with Onchocerca volvulus in a mouse model

Human onchocerciasis is a neglected tropical disease caused by Onchocerca volvulus and an important cause of blindness and chronic disability in the developing world. Although mass drug administration of ivermectin has had a profound effect on control of the disease, additional tools are critically needed including the need for a vaccine against onchocerciasis. The objectives of the present study were to: (i) select antigens with known vaccine pedigrees as components of a vaccine; (ii) produce the selected vaccine antigens under controlled conditions, using two expression systems and in one laboratory and (iii) evaluate their vaccine efficacy using a single immunisation protocol in mice. In addition, we tested the hypothesis that joining protective antigens as a fusion protein or in combination, into a multivalent vaccine, would improve the ability of the vaccine to induce protective immunity. Out of eight vaccine candidates tested in this study, Ov-103, Ov-RAL-2 and Ov-CPI-2M were shown to reproducibly induce protective immunity when administered individually, as fusion proteins or in combination. Although there was no increase in the level of protective immunity induced by combining the antigens into one vaccine, these antigens remain strong candidates for inclusion in a vaccine to control onchocerciasis in humans.

Soluble extract from the nematode Strongyloides stercoralis induces CXCR2 dependent/IL-17 independent neutrophil recruitment

Neutrophil recruitment via CXCR2 is required for innate and adaptive protective immunity to the larvae of Strongyloides stercoralis in mice. The goal of the present study was to determine the mechanism of CXCR2-mediated neutrophil recruitment to S. stercoralis. Mice deficient in the receptor for IL-17A and IL-17F, upstream mediators of CXCR2 ligand production, were infected with S. stercoralis larvae; there was no difference in larval survival, neutrophil recruitment, or production of CXCR2 ligands compared with wild type mice. In vivo and in vitro stimulation of neutrophils with S. stercoralis soluble extract resulted in significant neutrophil recruitment. In vitro assays demonstrated that the recruitment functioned through both chemokinesis and chemotaxis, was specific for CXCR2, and was a G protein-coupled response involving tyrosine kinase and PI3K. Finally, neutrophil stimulation with S. stercoralis soluble extract induced release of the CXCR2 ligands MIP-2 and KC from neutrophils, thereby potentially enhancing neutrophil recruitment.

Immunization with the recombinant antigen Ss-IR induces protective immunity to infection with Strongyloides stercoralis in mice

Human intestinal infections with the nematode Strongyloides stercoralis remain a significant problem worldwide and a vaccine would be a useful addition to the tools available to prevent and control this infection. The goal of this study was to test single antigens for their efficacy in a vaccine against S. stercoralis larvae in mice. Alum was used as the adjuvant in these studies and antigens selected for analysis were either recognized by protective human IgG (Ss-TMY-1, Ss-EAT-6, and Ss-LEC-5) or were known to be highly immunogenic in humans (Ss-NIE-1 and Ss-IR). Only mice immunized with the Ss-IR antigen demonstrated a significant decrease of approximately 80% in the survival of larval parasites in the challenge infection. Antibodies, recovered from mice with protective immunity to S. stercoralis after immunization with Ss-IR, were used to locate the antigen in the larvae. Confocal microscopy revealed that IgG from mice immunized with Ss-IR bound to the surface of the parasites and observations by electron microscopy indicated that IgG bound to granules in the glandular esophagus. Serum collected from mice immunized with Ss-IR passively transferred immunity to naïve mice. These studies demonstrate that Ss-IR, in combination with alum, induces high levels of protective immunity through an antibody dependent mechanism and may therefore be suitable for further development as a vaccine against human strongyloidiasis.