Thomas J. Nolan

Eosinophils Can Function as Antigen-Presenting Cells To Induce Primary and Secondary Immune Responses to Strongyloides stercoralis

ABSTRACT Several studies have demonstrated roles for eosinophils during innate and adaptive immune responses to helminth infections. However, evidence that eosinophils are capable of initiating an immune response to parasite antigens is lacking. The goal of the present in vitro study was to investigate the potential of eosinophils to serve as antigen-presenting cells (APC) and initiate an immune response to parasite antigens. Purified eosinophils were exposed to soluble Strongyloides stercoralis antigens, and the expression of various surface markers involved in cell activation was examined. Antigen-exposed eosinophils showed a sixfold increase in expression levels of CD69 and major histocompatibility complex (MHC) class II, a fourfold increase in levels of T-cell costimulatory molecule CD86, and a twofold decrease in levels of CD62L compared to eosinophils cultured in medium containing granulocyte-macrophage colony-stimulating factor. The ability of eosinophils to present antigen to T cells was determined by culturing them with T cells in vitro. Eosinophils pulsed with antigen stimulated antigen-specific primed T cells and CD4+ T cells to increase interleukin-5 (IL-5) production. The blocking of MHC class II expression on eosinophils inhibited their ability to induce IL-5 production by CD4+ T cells in culture. Antigen-pulsed eosinophils were able to prime naïve T cells and CD4+ T cells in culture and polarized them into Th2 cells producing IL-5 similar to that induced by antigen-loaded dendritic cells. These results demonstrate that eosinophils are capable of activating antigen-specific Th2 cells inducing the release of cytokines and assist in the priming of naïve T cells to initiate Th2 responses against infection. This study highlights the potential of eosinophils to actively induce immune responses against infection by amplifying antigen-specific Th2-cell responses.

Role of IL-5 in Innate and Adaptive Immunity to Larval Strongyloides stercoralis in Mice

Protective immunity to Strongyloides stercoralis infective larvae in mice has been shown to be dependent on IL-5 based on mAb depletion studies. The goal of this study was to determine the functional role of IL-5 during the innate and adaptive immune response to larval S. stercoralis in mice. In these studies, three strains of mice were used: wild-type C57BL/6J (WT), IL-5 knockout (KO), and IL-5 transgenic (TG). Innate responses to the larvae indicated that there was enhanced survival in the KO animals and decreased survival in the TG animals compared with WT. Furthermore, killing of larvae in TG mice was associated with eosinophil infiltration and degranulation. In studying the adaptive immune response, it was observed that immunization of KO mice did not lead to the development of protective immunity. Experiments were then performed to determine whether KO mice reconstituted with Abs or cells could then develop protective immunity. KO mice displayed protective immunity via a granulocyte-dependent mechanism following injection of purified IgM from immune wild-type animals. Immunity in KO mice could also be reconstituted by the injection of eosinophils at the time of immunization. These eosinophils did not participate in actively killing the challenge infection, but rather were responsible for the induction of a protective Ab response. We conclude that IL-5 is required in the protective immune response for the production of eosinophils, and that eosinophils were involved in larval killing during innate immunity and in the induction of protective Abs in the adaptive immune response.

Endoparasite prevalence and recurrence across different age groups of dogs and cats

The apparent prevalence of endoparasite infections across different age groups was calculated from 6555 dogs and 1566 cats that had a fecal examination performed upon presentation to the Veterinary Hospital of the University of Pennsylvania between 1997 and 2007. Based on notations from the medical history indicating prior parasite infections, estimates of recurrence were generated for each common group of parasites, including Trichuris, Giardia, ascarids, hookworms, Cystoisospora, and tapeworms. Endoparasitism was predominantly a disease of younger animals, with peak prevalence observed almost uniformly in dogs under 6 months old, with the exception of Trichuris with its longer pre-patent period, and in cats less than 18 months old. Furthermore, nearly 50% of dogs under 6 months old with a history of parasites, were diagnosed with at least one species of parasite on subsequent fecal examination. The percentage dropped to 18.4% in animals aged 1-4 years, but again increased to 31.5% in animals over 10 years old. There was no reported recurrence of Giardia or Cystoisospora from canine or feline patients older than 1 year. The recurrence of whipworm rose steadily with age, while hookworm and roundworm recurrence peaked in patients 1-4 years old. Findings from the study emphasize the importance of follow up fecal examinations and treatments in patients diagnosed with endoparasites.

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.

Time series analysis of the prevalence of endoparasitic infections in cats and dogs presented to a veterinary teaching hospital

The apparent prevalence of endoparasitic infections of cats and dogs presented to the small animal Veterinary Hospital of the University of Pennsylvania was measured between 1984 and 1991. Two thousand feline and 8077 canine fecal samples were examined along with 6830 canine blood samples. The overall mean monthly prevalence of feline infections was 16% for ascarids, 0.9% for hookworms, 4.0% for tapeworms, 2.4% for Giardia spp. and 4.2% for coccidia. The overall mean monthly prevalence of canine infections was 5.7% for ascarids, 9.7% for hookworms, 9.7% for whipworms. 1.8% for tapeworms, 4.7% for Giardia spp. and 3.1% for coccidia. There was a significant downward trend in the prevalence of hookworms and heartworms in dogs (P < 0.001 in both cases). There was a significant upward trend in the prevalence of tapeworms in cats (P < 0.05). There were no significant long-term trends in any of the other time series. The smoothed data were analyzed for seasonal trends. None of the autocorrelation analyses gave incontrovertible evidence of seasonality. The repeated peaks at the 6, 12 and 24 month lags in the case of ascarid infections were suggestive of a 12 month seasonality with a peak prevalence in December, but the results were not statistically significant at the 5% level. Hookworms and whipworms in dogs occurred together more than would be expected by chance in 4 out of the 6 years for which data were available.

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.

The role of B cells in immunity against larval Strongyloides stercoralis in mice

The objective of this study was to examine the role of B cells in primary and challenge infections of larval Strongyloides stercoralis in mice. Two strains of B‐cell deficient mice were used in these studies, µMT mice that lack all B cells and Xid mice that lack B‐1 cells. Primary immune responses in µMT mice were sufficient to eliminate all parasites within 1 week after infection. Immunized µMT and Xid mice, however, were unable to kill challenge parasites at 24 h post infection, the time that they were eliminated in immunized wild‐type mice. This was despite having a significant increase in interleukin‐5 secreting cells and high numbers of eosinophils in the microenvironment of the challenge larvae. In addition, immunized Xid mice did not generate parasite‐specific immunoglobulin (Ig)M but did develop a weak IgG response compared to wild‐type mice. These results demonstrate a dichotomy in the requirement of B cells in immunity to S. stercoralis. B cells are not required in the primary response, yet they are required in the secondary immune response. B‐1 cells are required for the secondary immune response and their role appears to be the production of IgM and not as a source of immunoregulatory molecules.

Specificity and mechanism of immunoglobulin M (IgM)- and IgG-dependent protective immunity to larval Strongyloides stercoralis in mice.

ABSTRACT Protective immunity in mice to the infective third-stage larvae (L3) of Strongyloides stercoralis was shown to be dependent on immunoglobulin M (IgM), complement activation, and granulocytes. The objectives of the present study were to determine whether IgG was also a protective antibody isotype and to define the specificity and the mechanism by which IgG functions. Purified IgG recovered from mice 3 weeks after a booster immunization with live L3 was shown to transfer high levels of protective immunity to naïve mice. IgG transferred into mice treated to block complement activation or to eliminate granulocytes failed to kill the challenge larvae. Transfer of immune IgG into IL-5 knockout (KO) mice, which are deficient in eosinophils, resulted in larval attrition, while transfer into FcRγ KO mice did not result in larval killing. These findings suggest that IgG from mice immunized with live L3 requires complement activation and neutrophils for killing of L3 through an antibody-dependent cellular cytotoxicity (ADCC) mechanism. This is in contrast to the results of investigations using IgM from mice immunized with live L3 and IgG from mice immunized with larval antigens soluble in deoxycholate in which protective immunity was shown to be ADCC independent. Western blot analyses with immune IgM and IgG identified few antigens recognized by all protective antibody isotypes. Results from immunoelectron microscopy demonstrated that the protective antibodies bound to different regions in the L3. It was therefore concluded that while IgM and IgG antibodies are both protective against larval S. stercoralis, they recognize different antigens and utilize different killing mechanisms.

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.