Browsing by Autor "Augusto Manubens"

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Hemocyanins Stimulate Innate Immunity by Inducing Different Temporal Patterns of Proinflammatory Cytokine Expression in Macrophages
(American Association of Immunologists, 2016) Ta-Ying Zhong; Sergio Arancibia; Raimundo Born; Robert Tampé; Javiera Villar; Miguel Del Campo; Augusto Manubens; Marı́a Inés Becker
Hemocyanins induce a potent Th1-dominant immune response with beneficial clinical outcomes when used as a carrier/adjuvant in vaccines and nonspecific immunostimulant in cancer. However, the mechanisms by which hemocyanins trigger innate immune responses, leading to beneficial adaptive immune responses, are unknown. This response is triggered by a proinflammatory signal from various components, of which macrophages are an essential part. To understand how these proteins influence macrophage response, we investigated the effects of mollusks hemocyanins with varying structural and immunological properties, including hemocyanins from Concholepas concholepas, Fissurella latimarginata, and Megathura crenulata (keyhole limpet hemocyanin), on cultures of peritoneal macrophages. Hemocyanins were phagocytosed and slowly processed. Analysis of this process showed differential gene expression along with protein levels of proinflammatory markers, including IL-1β, IL-6, IL-12p40, and TNF-α. An extended expression analysis of 84 cytokines during a 24-h period showed a robust proinflammatory response for F. latimarginata hemocyanin in comparison with keyhole limpet hemocyanin and C. concholepas hemocyanin, which was characterized by an increase in the transcript levels of M1 cytokines involved in leukocyte recruitment. These cytokine genes included chemokines (Cxcl1, Cxcl3, Cxcl5, Ccl2, and Ccl3), ILs (Il1b and Ifng), growth factors (Csf2 and Csf3), and TNF family members (Cd40lg). The protein levels of certain cytokines were increased. However, every hemocyanin maintains downregulated key M2 cytokine genes, including Il4 and Il5 Collectively, our data demonstrate that hemocyanins are able to trigger the release of proinflammatory factors with different patterns of cytokine expression, suggesting differential signaling pathways and transcriptional network mechanisms that lead to the activation of M1-polarized macrophages.
Macrophages stimulated with mollusk hemocyanins present a different expression pattern of proinflammatory cytokines and chemokines (VAC3P.956)
(American Association of Immunologists, 2014) Marı́a Inés Becker; Ta-Ying Zhong; Sergio Arancibia; Raimundo Born; Robert Tampé; Miguel Del Campo; Augusto Manubens
Abstract Hemocyanins inoculated in mammals induce a Th1-dominant response with beneficial clinical outcomes, being used as carrier/adjuvant in vaccines and as immunostimulant for the treatment of bladder cancer. However, its effects during the early phases of the immune response are unknown. We hypothesized that when hemocyanins are incorporated by antigen presenting cells, they induce an inflammatory milieu, producing a bystander effect, key event in its non-specific immunostimulatory effects. Here, in vitro cultures of murine peritoneal macrophages were incubated with different hemocyanins from M. crenulata (KLH) or C. concholepas (CCH) or F. latimarginata (FLH), to study the correlation between mRNA and protein levels from a panel of proinflammatory cytokines and chemokines, using qPCR and ELISA. The results showed that hemocyanins were endocyted and slowly processed by macrophages, stimulating the expression of IL-6, IL-12p40 and TNF-α at different times and intensities. FLH and KLH but not CCH at 24 h, induced an increased in the expression of chemokines related to leukocyte extravasations, such as CCL-3, CXCL-1 and CXCL-5. Moreover, hemocyanins kept down-regulated key Th2 cytokines such as IL-4 and TGF-β2. We concluded that each hemocyanin can stimulate different kinetic patterns of proinflammatory cytokines and chemokines expression in macrophages, suggesting different signaling pathways, which can be explained in part, by its structural differences.
Mechanisms underlying the mollusk hemocyanin processing and presentation through MHC-dependent pathways in antigen presenting cells of mammals
(American Association of Immunologists, 2022) Marı́a Inés Becker; Michelle L. Salazar; Diego A. Díaz-Dinamarca; Abel E. Vásquez; Javiera Villar; Alejandra Alvarado; Byron Castillo; Daniel Navarro; Fabián Salazar; Augusto Manubens
Abstract Hemocyanins are oligomeric glycoproteins widely used as immunomodulators because they bias immunity towards a Th1 profile when inoculated in mammals. We have demonstrated that hemocyanins are internalized through receptor-mediated endocytosis, and TLR4 and C-type lectin receptors (MR, DC-SIGN, MGL) participate in the hemocyanin-mediated proinflammatory response in mouse and human dendritic cells (DCs). However, despite the massive use of hemocyanins, their intracellular processing route for MHC presentation to T lymphocytes has been scarcely studied. Therefore, we hypothesized that hemocyanins follow the MHC-II pathway as a classical T-cell-dependent antigen. Interestingly, our results analyzing the processing pathway of hemocyanins in mouse DCs showed that hemocyanins from Fissurella latimarginata (FLH) and Megathura crenulata (KLH) co-localized with Rab5+, Rab7+, and Lamp-1+ compartments. This observation strongly suggests that hemocyanins could be cross-presented by MHC-I molecules. Furthermore, DCs incubated with FLH showed an increase in the percentage of MHC-I+ cells versus the control cells. FLH-induced cytokine secretion decreased in J774.2 macrophages treated with pharmacological inhibitors of both MHC-II and MHC-I pathways, supporting our previous results on hemocyanin cross-presentation but also the MHC-II pathway. Furthermore, immunoblot confirmed different FLH proteolysis patterns in macrophages treated with MHC-I and MHC-II pathway inhibitors. Hence, we postulate that hemocyanins undergo both MHC-I and MHC-II dependent antigen presentation pathways in antigen-presenting cells. These findings offer molecular clues to underlying hemocyanin processing and presentation mechanisms. Supported by grants from FONDECYT N° 1151337 and N° 1201600 (MIB), ANID/Beca Doctorado Nacional N° 21210946 (MLS) and N° 21200880 (DDD).
Novel hemocyanin from the Fissurella latimarginata exhibits an outstanding immunogenicity and non-specific immunomodulatory effects in a melanoma model (53.6)
(American Association of Immunologists, 2012) Marı́a Inés Becker; Sergio Arancibia; Espinoza Cecilia; Fabián Salazar; Miguel Del Campo; Raimundo Born; Jorge Ferreira; Augusto Manubens; Alfredo De Ioannes
Abstract The versatile properties of mollusk hemocyanins in biomedical applications, including non-specific immunostimulant during the therapy of bladder cancer and carrier/adjuvant in cutting-edge therapeutic cancer vaccines, has increased the interest in finding new hemocyanins with better immunomodulatory properties. Here, we evaluate the physicochemical and immunological properties of the hemocyanin from Fissurella latimarginata (FLH). This protein shares with keyhole limpet hemocyanin (KLH) and Concholepas hemocyanin (CCH) the typical hollow cylinder structure and it is also made by two subunits (~350 KDa). ConA lectin staining of FLH, demonstrated the presence of mannosylated carbohydrate structures as well as in KLH and CCH. The humoral responses in mice demonstrated that antibody titers induced by FLH itself were significantly higher than KLH and CCH. However, it performance as a carrier protein to induce antibodies to the hapten DNFB, was similar to the other carriers using Freund’s adjuvant as a repository. More interestingly, FLH exhibits an outstanding antitumor activity prolonging mice survival, when was evaluated and compared with KLH and CCH in the B16F10 mouse melanoma model. In addition, flow cytometry analysis showed that FLH induced a significant number of tumor-infiltrating CD4+ cells and also, IFN-δ secretion. Together, these findings introduce a novel hemocyanin, with intrinsically more immunogenic and immunomodulatory capacities for antitumor therapy.
Recombinant Surface Immunogenic Protein from <i>S. agalactiae</i> and hemocyanin from <i>Fisurella latimarginata</i> promote cross-presentation dependence on the TLR-4 signaling
(American Association of Immunologists, 2023) Diego A. Díaz-Dinamarca; Michelle Salazar; Augusto Manubens; Fabián Salazar; Mayarling F. Troncoso; Sergio Lavandero; Janepsy Díaz; Abel E. Vásquez; Marı́a Inés Becker
Abstract Improved vaccine adjuvants are needed to address infectious diseases, cancer, and future pandemics. Toll-like receptors (TLRs) have been researched as targets of vaccine adjuvants because they modulate the innate immune system and activate adaptive immunity. TLR-based adjuvants induce cross-presentation of exogenous antigens to CD8 T lymphocytes. TLR4 signaling recruits the adapters myeloid differentiation marker 88 (MyD88), which mediates the recycling of endoplasmic reticulum fusion to the phagosome as well as the TIR-domain-containing adapter-inducing interferon-β (TRIF), which is essential for translocation from the endosome to the cytosol. Given the role of TLR4 agonists, most studies have used ligands based on lipopolysaccharides, but these have side effects (especially fever). Here we propose using TLR4 protein-based agonists (TLR-4-PBA), which have multiple advantages as adjuvants: They are naturally biocompatible and biodegradable substances that induce minimal reactogenicity and toxicity. We studied mollusk hemocyanin from Fissurella latimarginata (FLH) and the recombinant surface immunogenic protein from Streptococcus agalactiae (rSIP). These two proteins differ significantly in their structure, and we show Th1 immunomodulatory properties. Using mouse bone marrow-derived dendritic cells pulsed with ovoalbumin (OVA), we then showed that rSIP and FLH promote cross-presentation in OT-I CD8+ T lymphocytes. This effect depends on TLR4 and the recruitment of MyD88 and TRIF. Studies are in progress to evaluate IRF3 and NF-κ B activation. Our results illustrate how these TLR-4-PBAs could act as a clinically useful vaccine adjuvant for innate immunity and Th1 adaptive immune response. FONDECYT 1201600 (MIB); FONDEF ID21I10370 (AEV), Instituto de Salud Pública de Chile Funding (AEV, DD-D); ANID/PhD Nacional program 21210946 (MLS) and 21200880 (DD-D); FONDAP 15130011 (MT, SL).