Glycans from Fasciola hepatica modulate the host immune response and TLR-Induced maturation of dendritic cells

Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.Fil: Rodriguez, Ernesto. Universidad de la República; UruguayFil: Noya, Verónica. Universidad de la República; UruguayFil: Cervi, Laura Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Chiribao, Maria Laura. Universidad de la República; UruguayFil: Brossard, Natalie. Universidad de la República; UruguayFil: Chiale, Carolina. Universidad de la República; UruguayFil: Carmona, Carlos. Universidad de la República; UruguayFil: Giacomini, Cecilia. Universidad de la República; UruguayFil: Freire, Teresa. Universidad de la República; Urugua

Modulation of Dendritic Cell Maturation by Fasciola hepatica: Implications of Glycans and Mucins for Vaccine Development

Fasciola hepatica is a worldwide distributed helminth pathogen that causes great economic losses in sheep andcattle. This parasite is able to regulate the host immune response, producing high levels of IL-5 and low levels ofIFNγ, as well as modulating the function of dendritic cells (DCs), mast cells or macrophages, among others.Moreover, TLR-mediated maturation of DCs can be suppressed by F. hepatica derived components. Here, weinvestigated the role of glycans in the modulation of LPS-induced maturation of DCs, as well as in the production ofIL-5 and IFNγ by splenocytes from infected mice. We show that F. hepatica induces the recruitment to theperitoneum of semi-matured DCs, as judged by a down-regulation of MHC class II molecule expression and anincrease of CD80 and CD86 expression of DCs in the peritoneum of infected animals. Furthermore, we provideevidence indicating that glycan structures from F. hepatica are responsible, at least in part, for inhibiting LPS-induced DC maturation and production of IFNγ by splenocytes from infected animals. On the other hand, we showthat a mucin-like non-glycosylated peptide highly expressed in NEJ (Fhmuc) is able to synergize with LPS ininducing DC-maturation, and that it induces a T cell response specific for F. hepatica, both alone or in combinationwith DCs. Our data highlight the role of F. hepatica glycans in modulating the host immune response and mightcontribute to the design of vaccines against fasciolosis.Fil: Noya, Veronica. Universidad de la República; UruguayFil: Rodriguez, Ernesto. Universidad de la República; UruguayFil: Cervi, Laura Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Giacomini, Cecilia. Universidad de la República; UruguayFil: Brossard, Natalie. Universidad de la República; UruguayFil: Chiale, Carolina. Universidad de la República; UruguayFil: Carmona, Carlos. Universidad de la República; UruguayFil: Freire, Teresa. Universidad de la República; Urugua

Human hydatid cyst fluid-induced therapeutic anti-cancer immune responses via NK1.1+ cell activation in mice

Echinococcus granulosus is a cestode parasite which causes cystic echinococcosis disease. Previously we observed that vaccination with E. granulosus antigens from human hydatid cyst fluid (HCF) significantly inhibits colon cancer growth. In the present work, we evaluate the anti-tumor immune response induced by human HCF against LL/2 lung cancer in mice. HCF vaccination protected from tumor growth, both in prophylactic and therapeutic settings, and significantly increased mouse survival compared to control mice. Considering that tumor-associated carbohydrate antigens are expressed in E. granulosus, we oxidized terminal carbohydrates in HCF with sodium periodate. This treatment abrogates the anti-tumor activity induced by HCF vaccination. We found that HCF vaccination-induced IgG antibodies that recognize LL/2 tumor cells by flow cytometry. An antigen-specific immune response is induced with HCF vaccination in the tumor-draining lymph nodes and spleen characterized by the production of IL-5 and, in less extent, IFNɣ. In the tumor microenvironment, we found that NK1.1 positive cells from HCF-treated mice showed higher expression of CD69 than control mice ones, indicating a higher level of activation. When we depleted these cells by administrating the NK-specific antibody NK1.1, a significantly decreased survival was observed in HCF-induced mice, suggesting that NK1.1+ cells mediate the anti-tumor protection induced by HCF. These results suggest that HCF can evoke an integrated anti-tumor immune response involving both, the innate and adaptive components, and provide novel insights into the understanding of the intricate relationship between HCF vaccination and tumor growth.Fil: Berriel, Edgardo. Instituto Pasteur de Montevideo; Uruguay. Hospital Pasteur Montevideo; UruguayFil: Freire Gard, Teresa Inés. Universidad de la Republica Facultad de Medicina; UruguayFil: Chiale, Carolina. Universidad de la Republica Facultad de Medicina; UruguayFil: Rodriguez, Ernesto Jorge. Universidad de la Republica Facultad de Medicina; UruguayFil: Moron, Victor Gabriel. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Fernández Graña, Gabriel. Instituto Pasteur de Montevideo; UruguayFil: Crispo, Martina. Instituto Pasteur de Montevideo; UruguayFil: Berois, Nora. Instituto Pasteur de Montevideo; UruguayFil: Osinaga, Eduardo. Instituto Pasteur de Montevideo; Uruguay. Universidad de la Republica Facultad de Medicina; Urugua

Trypanosoma cruzi extracts elicit protective immune response against chemically induced colon and mammary cancers

Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, has anticancer effects mediated, at least in part, by parasite-derived products which inhibit growth of tumor cells. We investigated whether immunity to T. cruzi antigens could induce antitumor activity, using two rat models which reproduce human carcinogenesis: colon cancer induced by 1,2-dimethylhydrazine (DMH), and mammary cancer induced by N-nitroso-N-methylurea (NMU). We found that vaccination with T. cruzi epimastigote lysates strongly inhibits tumor development in both animal models. Rats immunized with T. cruzi antigens induce activation of both CD4(+) and CD8(+) T cells and splenocytes from these animals showed higher cytotoxic responses against tumors as compared to rats receiving adjuvant alone. Tumor-associated immune responses included increasing number of CD11b/c(+) His48(-) MHC II(+) cells corresponding to macrophages and/or dendritic cells, which exhibited augmented NADPH-oxidase activity. We also found that T. cruzi lysate vaccination developed antibodies specific for colon and mammary rat cancer cells, which were capable of mediating antibody-dependent cellular cytotoxicity (ADCC) in vitro. Anti-T. cruzi antibodies cross-reacted with human colon and breast cancer cell lines and recognized 41/60 (68%) colon cancer and 38/63 (60%) breast cancer samples in a series of 123 human tumors. Our results suggest that T. cruzi antigens can evoke an integrated antitumor response involving both the cellular and humoral components of the immune response and provide novel insights into the understanding of the intricate relationship between parasite infection and tumor growthFil: Ubillos, Lluis. Universidad de la República; UruguayFil: Freire, Teresa. Universidad de la República; UruguayFil: Berriel, Edgardo. Instituto Pasteur de Montevideo; Uruguay. Universidad de la República; UruguayFil: Chiribao, María Laura. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; UruguayFil: Chiale, Carolina. Universidad de la República; UruguayFil: Festari, María Florencia. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; UruguayFil: Medeiros, Andrea. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; UruguayFil: Mazal, Daniel. Universidad de la República; UruguayFil: Rondan, Mariella. Universidad de la República; UruguayFil: Bollati Fogolin, Mariela. Instituto Pasteur de Montevideo; UruguayFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Robello, Carlos. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; UruguayFil: Osinaga, Eduardo. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; Urugua

Mucin-like peptides from Echinococcus granulosus induce antitumor activity

International audienceThere is substantial evidence suggesting that certain parasites can have antitumor properties. We evaluated mucin peptides derived from the helminth Echinococcus granulosus (denominated Egmuc) as potential inducers of antitumor activity. We present data showing that Egmuc peptides were capable of inducing an increase of activated NK cells in the spleen of immunized mice, a fact that was correlated with the capacity of splenocytes to mediate killing of tumor cells. We demonstrated that Egmuc peptides enhance LPS-induced maturation of dendritic cells in vitro by increasing the production of IL-12p40p70 and IL-6 and that Egmuc-treated DCs may activate NK cells, as judged by an increased expression of CD69. This evidence may contribute to the design of tumor vaccines and open new horizons in the use of parasite-derived molecules in the fight against cancer

MUC5B silencing reduces chemo-resistance of MCF-7 breast tumor cells and impairs maturation of dendritic cells.

International audienceMucins participate in cancer progression by regulating cell growth, adhesion, signaling, apoptosis or chemo-resistance to drugs. The secreted mucin MUC5B, the major component of the respiratory tract mucus, is aberrantly expressed in breast cancer, where it could constitute a cancer biomarker. In this study we evaluated the role of MUC5B in breast cancer by gene silencing the MUC5B expression with short hairpin RNA on MCF-7 cells. We found that MUC5B-silenced MCF-7 cells have a reduced capacity to grow, adhere and form cell colonies. Interestingly, MUC5B knock-down increased the sensitivity to death induced by chemotherapeutic drugs. We also show that MUC5B silencing impaired LPS-maturation of DCs, and production of cytokines. Furthermore, MUC5B knock-down also influenced DC-differentiation and activation since it resulted in an upregulation of IL-1β, IL-6 and IL-10, cytokines that might be involved in cancer progression. Thus, MUC5B could enhance the production of LPS-induced cytokines, suggesting that the use of MUC5B-based cancer vaccines combined with DC-maturation stimuli, could favor the induction of an antitumor immune response

Oxidation of parasite components partially inhibits modulation of LPS-induced maturation of DCs by FhTE.

<p>BMDCs were cultured in the presence of 75 μg/mL of FhTE, FhCB (oxidation negative control) or FhmPox (oxidized FhTE) in presence or absence of LPS (1 μg/ml) overnight at 37°C. Then, culture supernatants were collected and analyzed by ELISA for IL-6, IL-10 or IL-12/23p40 (<b>A</b>). BMDCs were also pre-incubated for 45 min. with mannose (Man), N-Acetyl-Galactosamine (GalNAc) or arabinose (Ara) at 10 mM and then stimulated as in A. Culture supernatants were analyzed by ELISA for detection of IL-6, IL-10 or IL-12/23p40 (<b>B</b>) and MIP-1α and MIP-2 (<b>C</b>). Alternatively, BMDCs were pre-incubated for 45 min. with 10 μM of specific signaling inhibitors (PHPS1; GW5074; and ER27319) and then stimulated with FhTE (75 μg/mL) in presence of LPS (1 μg/ml) (<b>D</b>). Results are expressed as the mean of three independent experiments (±SD, indicated by error bars). Asterisks indicate statistically significant differences (*<i>p</i> < 0.01) with respect to LPS-stimulated BMDCs.</p

<i>F</i>. <i>hepatica</i> infection promotes the recruitment of MHC^low IL-10⁺ DCs both at the peritoneal cavity and spleen.

<p>Mice (n = 5 per group) were orally infected with 10 metacercariae in PBS (infected mice). PBS alone served as a control (non-infected mice). Mice were sacrificed one, two and three weeks after the infection and spleens and PECs were removed. Splenocyte (<b>A</b>) and PEC (<b>B</b>) suspensions were counted and the presence of CD11c^hi cells was analyzed by flow cytometry by staining cells with specific antibodies. CD11c^hi cells were selected after excluding CD3⁺ followed by exclusion of F4/80⁺ cells (C). Splenocytes (<b>D</b>) and PECs (<b>E</b>) were also incubated with anti-MCHII, permeabilized, and intracellularly stained with anti-IL-10 and IL-12/23p40 antibodies for 30 min at 4°C. Cells were analyzed on a flow cytometer. Results are expressed as the mean of three independent experiments (±SD, indicated by error bars). Asterisks indicate statistically significant differences (*<i>p</i> < 0.01) with respect to cells from non-infected animals.</p

<i>F</i>. <i>hepatica</i> produces a diverse variety of glycan structures.

<p><b>(A)</b> Lectin reactivity was evaluated on microplates coated with FhTE (2.5 μg/well) using different concentrations of biotin-conjugated lectins and streptavidin-Dylight-800. Mean fluorescence intensity (MFI) was determined on plates with an infrared imaging system. Lectins used in this study were: VV (GalNAc, Tn antigen), WGA ((GlcNAc)₂), ConA (αMan>αGlc), PNA (βGal(1–3)GalNAc) and UEA (Fucα(1–2)Gal), ECA (βGal(1–4)GlcNAc), SNA (αNeuAc(2–6)Gal) and HPM (GalNAc). <b>(B)</b> Carbohydrate specificity was demonstrated by performing inhibition assays with specific carbohydrates (50 mM) by pre-incubating with GalNAc (VV), GlcNAc (WGA), Man (ConA), Gal (PNA) or Fuc (UEA). Alternatively, lectin reactivity was evaluated on periodate-oxidized glycans (FhmPox) or control consisting of FhTE only treated with borydrure (FhCB). <b>(C)</b> Parasite lysates were subjected to SDS-PAGE (15%) and stained with silver nitrate. <b>(D)</b> Alternatively, they were transferred to PVDF membranes and incubated for 2 h at RT with the anti-Tn monoclonal antibody 83D4 or a polyclonal anti-cathepsin L1 serum.</p

BMDCs pulsed with oxidized parasite lysate induce a decreased production of IL-4 and IL-10 by specific CD4⁺ T cells.

<p>BMDCs were cultured in the presence of 75 μg/mL of FhTE, FhCB (oxidation negative control) or FhmPox (oxidized FhTE) in the presence of LPS (1 μg/ml) overnight at 37°C. Then cell viability was evaluated by incubating with MTT for 4 h and reading absorbance at 570 nm (<b>A</b>). Alternatively, BMDCs were washed twice in complete medium and co-cultured for 3 days at 37°C with total splenocytes (<b>B</b>) or purified CD4⁺ T cells (<b>C</b>) from infected animals sacrificed after 3 wpi. Culture supernatants were collected and analyzed by ELISA for IL-4, IL-5, IL-10 or IFNγ Results are expressed as the mean of three independent experiments (±SD, indicated by error bars). Asterisks indicate statistically significant differences (**<i>p</i> < 0.01; *<i>p</i> < 0.05) with respect to FhTE/LPS-stimulated DCs.</p