Early Peritoneal Immune Response during Echinococcus granulosus Establishment Displays a Biphasic Behavior

Cystic echinococcosis is a zoonotic disease caused by the larval stage of the cestode Echinococcus granulosus and shows a cosmopolitan distribution with a worldwide prevalence of roughly 6 million infected people. Human cystic echinococcosis can develop in two types of infection. Primary infection occurs by ingestion of oncospheres, while secondary infection is caused by dissemination of protoscoleces after accidental rupture of fertile cysts. Murine experimental secondary infection in Balb/c mice is the current model to study E. granulosus-host interaction. Secondary infection can be divided into two stages: an early stage in which protoscoleces develop into hydatid cysts (infection establishment) and a later stage in which already differentiated cysts grow and eventually become fertile cysts (chronic infection). During infection establishment parasites are more susceptible to immune attack, thus our study focused on the immunological phenomena triggered early in the peritoneal cavity of experimentally infected mice. Our results suggest that early and local Th2-type responses are permissive for infection establishment

The ectodomains of the lymphocyte scavenger receptors CD5 and CD6 interact with tegumental antigens from Echinococcus granulosus sensu lato and protect mice against secondary cystic echinococcosis

Background: Scavenger Receptors (SRs) from the host's innate immune system are known to bind multiple ligands to promote the removal of non-self or altered-self targets. CD5 and CD6 are two highly homologous class I SRs mainly expressed on all T cells and the B1a cell subset, and involved in the fine tuning of activation and differentiation signals delivered by the antigen-specific receptors (TCR and BCR, respectively), to which they physically associate. Additionally, CD5 and CD6 have been shown to interact with and sense the presence of conserved pathogen-associated structures from bacteria, fungi and/or viruses. Methodology/Principal findings: We report herein the interaction of CD5 and CD6 lymphocyte surface receptors with Echinococcus granulosus sensu lato (s.l.). Binding studies show that both soluble and membrane-bound forms of CD5 and CD6 bind to intact viable protoscoleces from E. granulosus s.l. through recognition of metaperiodate-resistant tegumental components. Proteomic analyses allowed identification of thioredoxin peroxidase for CD5, and peptidyl-prolyl cis-trans isomerase (cyclophilin) and endophilin B1 (antigen P-29) for CD6, as their potential interactors. Further in vitro assays demonstrate that membrane-bound or soluble CD5 and CD6 forms differentially modulate the pro- and anti-inflammatory cytokine release induced following peritoneal cells exposure to E. granulosus s.l. tegumental components. Importantly, prophylactic infusion of soluble CD5 or CD6 significantly ameliorated the infection outcome in the mouse model of secondary cystic echinococcosis. Conclusions/Significance Taken together, the results expand the pathogen binding properties of CD5 and CD6 and provide novel evidence for their therapeutic potential in human cystic echinococcosis. Author summary: Scavenger Receptors (SRs) are constituents of host's innate immune system able to sense and remove altered-self and/or pathogen components. Data on their interaction with helminth parasites is scarce. In this work, we describe that CD5 and CD6 -two lymphoid SRs previously reported to interact with conserved structures from bacteria, fungi and viruses- recognize tegumental components in the cestode parasite Echinococcus granulosus sensu lato (s.l.). Moreover, both receptors differentially modulate the cytokine release by host cells exposed to E. granulosus s.l. tegumental components. Importantly, the infusion of soluble forms of CD5 or CD6 improve infection outcomes in a murine model of secondary cystic echinococcosis. In summary, our results expand the pathogen binding properties of CD5 and CD6 and suggest their therapeutic potential against helminth infections

Natural and induced antibodies contribute to differential susceptibility to secondary cystic echinococcosis of Balb/c and C57Bl/6 mice

Antibodies are key immune players in several helminth infections and animal models have been central for the identification of their mechanisms of protection. Murine secondary cystic echinococcosis is a useful model for studying Echinococcus granulosus immunobiology, being the immune profile mounted by the experimental host a determinant of parasite success or failure in infection establishment. In the present study, we analyzed infection outcome using Balb/c and C57Bl/6 mice strains, and compared their antibody responses in terms of quality and intensity. Our results showed that Balb/c is a highly susceptible strain to secondary cystic echinococcosis, while C57Bl/6 mice are quite resistant. Moreover, significant differences between strains were observed in natural and induced antibodies recognizing E. granulosus antigens, both at the systemic and peritoneal levels. Natural cross-reacting IgM, IgG2b and IgG3 antibodies were detected in sera from both strains but with different intensities, and - remarkably - natural IgG2b showed to be an intrinsic correlate of protection in both mice strains. Interestingly, naïve C57Bl/6 serum displayed a higher protoscolicidal activity, and heterologous - but not homologous - transference of C57Bl/6 naïve serum into Balb/c mice, significantly reduced their infection susceptibility. In the peritoneal cavity, different levels of natural cross-reacting IgM and IgG3 antibodies were detected in both mice strains, while cross-reacting IgG2b was detected only in C57Bl/6 mice. On the other hand, infected mice from both strains developed isotype-mixed antibody responses, with Balb/c mice biasing their response towards high avidity IgG1 and C57Bl/6 mice showing a predominance of mixed IgM/IgG2c/IgG2b/IgG3. In this regard, IgG1 levels showed to be a correlate of susceptibility in both mice strains. In conclusion, our results suggest that antibodies - either natural or induced - play a role in the susceptibility degree to murine secondary cystic echinococcosis.Fil: Mourglia Ettlin, Gustavo. Universidad de la República; UruguayFil: Cucher, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Arbildi, Paula. Universidad de la República; UruguayFil: Rosenzvit, Mara Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Dematteis, Sylvia. Universidad de la República; Urugua

Isolation and Characterization of a Secretory Component of Echinococcus multilocularis Metacestodes Potentially Involved in Modulating the Host-Parasite Interface

Echinococcus multilocularis metacestodes are fluid-filled, vesicle-like organisms, which are characterized by continuous asexual proliferation via external budding of daughter vesicles, predominantly in the livers of infected individuals. Tumor-like growth eventually leads to the disease alveolar echinococcosis (AE). We employed the monoclonal antibody (MAb) E492/G1, previously shown to be directed against a carbohydrate-rich, immunomodulatory fraction of Echinococcus granulosus, to characterize potentially related components in E. multilocularis. Immunofluorescence studies demonstrated that MAb E492/G1-reactive epitopes were found predominantly on the laminated layer and in the periphery of developing brood capsules. The respective molecules were continuously released into the exterior medium and were also found in the parasite vesicle fluid. The MAb E492/G1-reactive fraction in E. multilocularis, named Em492 antigen, was isolated by immunoaffinity chromatography. Em492 antigen had a protein/carbohydrate ratio of 0.25, reacted with a series of lectins, and is related to the laminated layer-associated Em2(G11) antigen. The epitope recognized by MAb E492/G1 was sensitive to sodium periodate but was not affected by protease treatment. Anti-Em492 immunoglobulin G1 (IgG1) and IgG2 and, at lower levels, IgG3 were found in sera of mice suffering from experimentally induced secondary, but not primary, AE. However, with regard to cellular immunity, a suppressive effect on concanavalin A- or crude parasite extract-induced splenocyte proliferation in these mice was observed upon addition of Em492 antigen, but trypan blue exclusion tests and transmission electron microscopy failed to reveal any cytotoxic effect in Em492 antigen-treated spleen cells. This indicated that Em492 antigen could be modulating the periparasitic cellular environment during E. multilocularis infection through as yet unidentified mechanisms and could be one of the factors contributing to immunosuppressive events that occur at the host-parasite interface

Early immunological phenomena in the peritoneal cavity of infected mice.

<p>After ip inoculation of protoscoleces into Balb/c mice approximately only 10% of parasites finally develop into hydatid cysts. Therefore, early immune responses although not optimal to prevent infection establishment, are probably not entirely ineffective. Based on our results, we suggest that NK cells may play an important role in very early infection. Activated NK cells through IFN-γ production could be partially responsible for the activation of resident macrophages inducing their protoscolicidal activity. IFNγ-activated macrophages as well as local antibodies recognizing protoscolex antigens and able to activate the complement system could be partially responsible for the elimination of 90% of parasite inoculum. From day 5 pi <i>E. granulosus</i> protoscoleces would bias the cytokine response towards a Th2-type profile and induce/recruit cells with regulatory activity (Treg). These phenomena would block initially triggered effects by IFN-γ. Moreover, antibody isotype restriction (IgG2b and IgM) could be seen as a possible evasion strategy exploited by <i>E. granulosus</i> since protoscoleces have Fc-like receptors able to bind specific isotypes trough their Fc portions. Overall, this schematic representation shows a possible explanation for the reasons of infection establishment in mice.</p

Peritoneal B cells drop is associated with a plasma cell differentiation process.

<p>A group of mice (n = 12) was inoculated ip with 2000 protoscoleces and another group (n = 8) was inoculated with equal volume of sterile PBS (control group). Three infected and 2 control animals were sacrificed at days 3, 5, 7 and 9 pi and their peritoneal cells were recovered. qRT-PCR was performed using specific primers for murine Pax5, Blimp-1 and Bcl-6, and relative mRNA levels were expressed respect to control group. Results are shown as group median and data range (A and B). Another group of infected (n = 6) and control mice (n = 6) were sacrificed at day 5 pi and their peritoneal leukocytes were culture for 72 h in complete RPMI without stimulation. Anti-PSA specific IgM, IgG1, IgG2a, IgG2b and IgG3 titers were determined by ELISA in culture supernatants. Results are shown as individual values (circles) and group median (lines) (C and D). (*) Statistical significance (p<0.05) compared to control group. Results are representative of two independent experiments.</p

Peritoneal cytokine response shows an early biphasic behavior.

<p>A group of mice (n = 12) was inoculated ip with 2000 protoscoleces and another group (n = 8) was inoculated with equal volume of sterile PBS (control group). Three infected and 2 control animals were sacrificed at days 3, 5, 7 and 9 pi and their peritoneal cells were recovered. qRT-PCR was performed using specific primers for murine IL-2 (A), IFN-γ (B), IL-15, IL-12 and TNFα (C), IL-6 (D), IL-4 and IL-13 (E), IL-5 and IL-10 (F). Relative mRNA levels are expressed respect to control group. Results are shown as group median and data range. (*) Statistical significance (p<0.05) compared to control group.</p

The ectodomains of the lymphocyte scavenger receptors CD5 and CD6 interact with tegumental antigens from Echinococcus granulosus sensu lato and protect mice against secondary cystic echinococcosis.

BackgroundScavenger Receptors (SRs) from the host's innate immune system are known to bind multiple ligands to promote the removal of non-self or altered-self targets. CD5 and CD6 are two highly homologous class I SRs mainly expressed on all T cells and the B1a cell subset, and involved in the fine tuning of activation and differentiation signals delivered by the antigen-specific receptors (TCR and BCR, respectively), to which they physically associate. Additionally, CD5 and CD6 have been shown to interact with and sense the presence of conserved pathogen-associated structures from bacteria, fungi and/or viruses.Methodology/principal findingsWe report herein the interaction of CD5 and CD6 lymphocyte surface receptors with Echinococcus granulosus sensu lato (s.l.). Binding studies show that both soluble and membrane-bound forms of CD5 and CD6 bind to intact viable protoscoleces from E. granulosus s.l. through recognition of metaperiodate-resistant tegumental components. Proteomic analyses allowed identification of thioredoxin peroxidase for CD5, and peptidyl-prolyl cis-trans isomerase (cyclophilin) and endophilin B1 (antigen P-29) for CD6, as their potential interactors. Further in vitro assays demonstrate that membrane-bound or soluble CD5 and CD6 forms differentially modulate the pro- and anti-inflammatory cytokine release induced following peritoneal cells exposure to E. granulosus s.l. tegumental components. Importantly, prophylactic infusion of soluble CD5 or CD6 significantly ameliorated the infection outcome in the mouse model of secondary cystic echinococcosis.Conclusions/significanceTaken together, the results expand the pathogen binding properties of CD5 and CD6 and provide novel evidence for their therapeutic potential in human cystic echinococcosis

Combining proteomics and bioinformatics to explore novel tegumental antigens as vaccine candidates against Echinococcus granulosus infection

Echinococcus granulosus is the parasite responsible for cystic echinococcosis (CE), an important worldwide-distributed zoonosis. New effective vaccines against CE could potentially have great economic and health benefits. Here, we describe an innovative vaccine design scheme starting from an antigenic fraction enriched in tegumental antigens from the protoscolex stage (termed PSEx) already known to induce protection against CE. We first used mass spectrometry to characterize the protein composition of PSEx followed by Gene Ontology analysis to study the potential Biological Processes, Molecular Functions, and Cellular Localizations of the identified proteins. Following, antigenicity predictions and determination of conservancy degree against other organisms were determined. Thus, nine novel proteins were identified as potential vaccine candidates. Furthermore, linear B cell epitopes free of posttranslational modifications were predicted in the whole PSEx proteome through colocalization of in silico predicted epitopes within peptide fragments identified by matrix-assisted laser desorption/ionization-TOF/TOF. Resulting peptides were termed “clean linear B cell epitopes,” and through BLASTp scanning against all nonhelminth proteins, those with 100% identity against any other protein were discarded. Then, the secondary structure was predicted for peptides and their corresponding proteins. Peptides with highly similar secondary structure respect to their parental protein were selected, and those potentially toxic and/or allergenic were discarded. Finally, the selected clean linear B cell epitopes were mapped within their corresponding 3D-modeled protein to analyze their possible antibody accessibilities, resulting in 14 putative peptide vaccine candidates. We propose nine novel proteins and 14 peptides to be further tested as vaccine candidates against CE.Fil: Miles, Sebastián. Universidad de la República; UruguayFil: Portela, Madelón. Instituto Pasteur de Montevideo; UruguayFil: Cyrklaff, Marek. Heidelberg University; AlemaniaFil: Ancarola, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Frischknecht, Friedrich. Heidelberg University; AlemaniaFil: Durán, Rosario. Instituto Pasteur de Montevideo; UruguayFil: Dematteis, Sylvia. Universidad de la República; UruguayFil: Mourglia Ettlin, Gustavo. Universidad de la República; Urugua