Early Immune Response Elicited by Different Trypanosoma cruzi Infective Stages

Trypanosoma cruzi is a protozoan parasite that affects millions of people in Latin America. Infection occurs by vectorial transmission or by transfusion or transplacental route. Immune events occurring immediately after the parasite entrance are poorly explored. Dendritic cells (DCs) are target for the parasite immune evasion mechanisms. Recently, we have demonstrated that two different populations of DCs display variable activation after interaction with the two infective forms of the parasite: metacyclic or blood trypomastigotes (mTp or bTp) in vitro. The skin constitutes a complex network with several populations of antigen-presenting cells. Previously, we have demonstrated T. cruzi conditioning the repertoire of cells recruited into the site of infection. In the present work, we observed that mTp and bTp inoculation displayed differences in cell recruitment to the site of infection and in the activation status of APCs in draining lymph nodes and spleen during acute infection. Animals inoculated with mTp exhibited 100% of survival with no detectable parasitemia, in contrast with those injected with bTp that displayed high mortality and high parasite load. Animals infected with mTp and challenged with a lethal dose of bTp 15 days after primary infection showed no mortality and incremented DC activation in secondary lymphoid organs compared with controls injected only with bTp or non-infected mice. These animals also displayed a smaller number of amastigote nests in cardiac tissue and more CD8 T cells than mice infected with bTp. All the results suggest that both Tp infective stages induce an unequal immune response since the beginning of the infection.Fil: Gutierrez, Brenda Celeste. 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: Lammel, Estela María. 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: González, Stella Maris. 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: Poncini, Carolina Verónica. 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; Argentin

Galectins in Chagas disease: A missing link between Trypanosoma cruzi infection, inflammation, and tissue damage

Trypanosoma cruzi, the protozoan parasite causative agent of Chagas disease, affects about seven million people worldwide, representing a major global public health concern with relevant socioeconomic consequences, particularly in developing countries. In this review, we discuss the multiple roles of galectins, a family of β-galactoside-binding proteins, in modulating both T. cruzi infection and immunoregulation. Specifically, we focus on galectin-driven circuits that link parasite invasion and inflammation and reprogram innate and adaptive immune responses. Understanding the dynamics of galectins and their β-galactoside-specific ligands during the pathogenesis of T. cruzi infection and elucidating their roles in immunoregulation, inflammation, and tissue damage offer new rational opportunities for treating this devastating neglected disease.Fil: Poncini, Carolina Verónica. 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: Benatar, Alejandro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: 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; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin

TNFR1 signaling contributes to T cell anergy during staphylococcus aureus sepsis

Early research on sepsis has focused on the initial hyper-inflammatory, cytokine mediated phase of the disorder whereas the events that govern the concomitant and subsequent anti-inflammatory compensatory response are not completely understood. In this context, the putative participation of TNFR1-mediated signaling in the immunosuppressive phase of Staphylococcus aureus sepsis has not been elucidated. The aim of this study was to determine the role of TNFR1 in directing the immune dysfunction during S. aureus sepsis and the potential contribution of MDSC to this process. Using a model of sepsis of peritoneal origin and tnfr1 -/- mice, we demonstrated that during staphylococcal sepsis CD4 + T cell anergy is significantly dependent on TNFR1 expression and that signaling through this receptor has an impact on bacterial clearance in the spleen. MDSC played a major role in the generation of anergic CD4 + T cells and their accumulation in the spleen during S. aureus sepsis correlated with IL-6 induction. Although TNFR1 signaling was not required for MDSC accumulation and expansion in the spleen, it determined the in vivo expression of Arginase 1 and iNOS, enzymes known to participate in the suppressive function of this population. Moreover, our data indicate that TNFR1-mediated IL-10 production may modulate MDSC function during staphylococcal sepsis. Taken together these results indicate that TNFR1 plays a critical role on T cell dysfunction during S. aureus sepsis by regulating immunomodulatory mediators in MDSC. The role of TNFR1-mediated signaling during the immunosuppressive phase of staphylococcal sepsis should be considered when designing novel alternative therapeutic approaches.Fil: Ledo, Camila. Universidad Maimónides; 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: Gonzalez, Cintia Daniela. 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: Poncini, Carolina Verónica. 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: Mollerach, Marta Eugenia. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gomez, Marisa Ines. Universidad Maimónides; 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; Argentin

Modulatory Effect of Trypanosoma cruzi Infective Stages in Different Dendritic Cell Populations in vitro

Trypanosoma cruzi is a protozoan parasite that infects at least 7 million persons in the world (OMS, 2019). In endemic areas, infection normally occurs by vectorial transmission; however, outside, it normally happens by blood and includes congenital transmission. The persistence of T. cruzi during infection suggests the presence of immune evasion mechanisms and the modulation of the anti-parasite response to a profile incapable of eradicating the parasite. Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells (APCs) that patrol tissues with a key role in mediating the interface between the innate and adaptive immune response. Previous results from our lab and other groups have demonstrated that T. cruzi modulates the functional properties of DCs, in vitro and in vivo. During vectorial transmission, metacyclic (m) trypomastigotes (Tps) eliminated along with the insect feces reach the mucous membranes or injured skin. When transmission occurs by the hematic route, the parasite stage involved in the infection is the circulating or blood (b) Tp. Here, we studied in vitro the effect of both infective mTp and bTp in two different populations of DCs, bone marrow–derived DCs (BMDCs) and XS106, a cell line derived from epidermal DCs. Results demonstrated that the interaction of both Tps imparts a different effect in the functionality of these two populations of DCs, suggesting that the stage of T. cruzi and DC maturation status could define the immune response from the beginning of the ingress of the parasite, conditioning the course of the infection.Fil: Gutierrez, Brenda Celeste. 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: Lammel, Estela María. 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: Ramirez, Marcel Ivan. Fundación Oswaldo Cruz; Brasil. Universidade Federal do Paraná; BrasilFil: González, Stella Maris. 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: Poncini, Carolina Verónica. 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; Argentin

Extracellular vesicles from Trypanosoma cruzi-dendritic cell interaction show modulatory properties and confer resistance to lethal infection as a cell-free based therapy strategy

Extracellular vesicles (EVs) include a heterogeneous group of particles. Microvesicles, apoptotic bodies and exosomes are the most characterized vesicles. They can be distinguished by their size, morphology, origin and molecular composition. To date, increasing studies demonstrate that EVs mediate intercellular communication. EVs reach considerable interest in the scientific community due to their role in diverse processes including antigenpresentation, stimulation of anti-tumoral immune responses, tolerogenic or inflammatory effects. In pathogens, EV shedding is well described in fungi, bacteria, protozoan and helminths parasites. For Trypanosoma cruzi EV liberation and protein composition was previously described. Dendritic cells (DCs), among other cells, are key players promoting the immune response against pathogens and also maintaining self-tolerance. In previous reports we have demonstrate that T. cruzi downregulates DCs immunogenicity in vitro and in vivo. Here we analyze EVs from the in vitro interaction between blood circulating trypomastigotes (Tp) and bone-marrow-derived DCs. We found that Tp incremented the number and the size of EVs in cultures with DCs. EVs displayed some exosome markers and intracellular RNA. Protein analysis demonstrated that the parasite changes the DC protein-EV profile. We observed that EVs from the interaction of Tp-DCs were easily captured by unstimulated-DCs in comparison with EVs from DCs cultured without the parasite, and also modified the activation status of LPS-stimulated DCs. Noteworthy, we found protection in animals treated with EVs-DCs+Tp and challenged with T. cruzi lethal infection. Our goal is to go deep into the molecular characterization of EVs from the DCs-Tp interaction, in order to identify mediators for therapeutic purposes

Targeting Myeloid-Derived Suppressor Cells to Enhance a Trans-Sialidase-Based Vaccine Against Trypanosoma cruzi

Trypanosoma cruzi (T. cruzi) is a hemoflagellate protozoan parasite that causes Chagas disease, a neglected tropical disease that affects more than 6 million people around the world, mostly in Latin America. Despite intensive research, there is no vaccine available; therefore, new approaches are needed to further improve vaccine efficacy. It is well established that experimental T. cruzi infection induces a marked immunosuppressed state, which includes notably increases of CD11b+ GR-1+ myeloid-derived suppressor cells (MDSCs) in the spleen, liver and heart of infected mice. We previously showed that a trans-sialidase based vaccine (TSf-ISPA) is able to confer protection against a virulent T. cruzi strain, stimulating the effector immune response and decreasing CD11b+ GR-1+ splenocytes significantly. Here, we show that even in the immunological context elicited by the TSf-ISPA vaccine, the remaining MDSCs are still able to influence several immune populations. Depletion of MDSCs with 5 fluorouracil (5FU) at day 15 post-infection notably reshaped the immune response, as evidenced by flow cytometry of spleen cells obtained from mice after 21 days post-infection. After infection, TSf-ISPA-vaccinated and 5FU-treated mice showed a marked increase of the CD8 response, which included an increased expression of CD107a and CD44 markers in CD8+ cultured splenocytes. In addition, vaccinated and MDSC depleted mice showed an increase in the percentage and number of CD4+ Foxp3+ regulatory T cells (Tregs) as well as in the expression of Foxp3+ in CD4+ splenocytes. Furthermore, depletion of MDSCs also caused changes in the percentage and number of CD11chigh CD8α+ dendritic cells as well as in activation/maturation markers such as CD80, CD40 and MHCII. Thus, the obtained results suggest that MDSCs not only play a role suppressing the effector response during T. cruzi infection, but also strongly modulate the immune response in vaccinated mice, even when the vaccine formulation has a significant protective capacity. Although MDSC depletion at day 15 post-infection did not ameliorated survival or parasitemia levels, depletion of MDSCs during the first week of infection caused a beneficial trend in parasitemia and mice survival of vaccinated mice, supporting the possibility to target MDSCs from different approaches to enhance vaccine efficacy. Finally, since we previously showed that TSf-ISPA immunization causes a slight but significant increase of CD11b+ GR-1+ splenocytes, here we also targeted those cells at the stage of immunization, prior to T. cruzi challenge. Notably, 5FU administration before each dose of TSf-ISPA vaccine was able to significantly ameliorate survival and decrease parasitemia levels of TSf-ISPA-vaccinated and infected mice. Overall, this work supports that targeting MDSCs may be a valuable tool during vaccine design against T. cruzi, and likely for other pathologies that are characterized by the subversion of the immune system.Fil: Gamba, Juan Cruz. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Tecnología Inmunológica; ArgentinaFil: Roldán, Carolina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Tecnología Inmunológica; ArgentinaFil: Prochetto, Estefanía Soledad. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Tecnología Inmunológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Lupi, Giuliana Antonella. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Tecnología Inmunológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Bontempi, Iván. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Tecnología Inmunológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Médicas; ArgentinaFil: Poncini, Carolina Verónica. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Cátedra de Microbiología, Parasitología e Inmunología; ArgentinaFil: Vermeulen, Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Perez, Ana Rosasanta fe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; ArgentinaFil: Marcipar, Iván Sergio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Tecnología Inmunológica; ArgentinaFil: Cabrera, Gabriel Gustavo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Tecnología Inmunológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin

Impairment in natural killer cells editing of immature dendritic cells by infection with a virulent Trypanosoma cruzi population

Early interactions between natural killer (NK) and dendritic cells (DC) shape the immune response at the frontier of innate and adaptive immunity. Activated NK cells participate in maturation or deletion of DCs that remain immature. We previously demonstrated that infection with a high virulence (HV) population of the protozoan parasite Trypanosoma cruzi downmodulates DC maturation and T-cell activation capacity. Here, we evaluated the role of NK cells in regulating the maturation level of DCs. Shortly after infection with HV T. cruzi, DCs in poor maturation status begin to accumulate in mouse spleen. Although infection induces NK cell cytotoxicity and cytokine production, NK cells from mice infected with HV T. cruzi exhibit reduced ability to lyse and fail to induce maturation of bone marrow-derived immature DCs (iDCs). NK-mediated lysis of iDCs is restored by in vitro blockade of the IL-10 receptor during NK-DC interaction or when NK cells are obtained from T. cruzi-infected IL-10 knockout mice. These results suggest that infection with a virulent T. cruzi strain alters NK cell-mediated regulation of the adaptive immune response induced by DCs. This regulatory circuit where IL-10 appears to participate might lead to parasite persistence but can also limit the induction of a vigorous tissue-damaging T-cell response.Fil: Batalla, Estela. 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: Pino Martínez, Agustina María. 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: Poncini, Carolina Verónica. 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: Duffy, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: González, Stella Maris. 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: Alba Soto, Catalina Dirney. 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; Argentin

Dendritic cell funtionality during Trypanosoma cruzi infection

Una de las características más relevantes de la infección por Trypanosoma cruzi es la presencia de desordenes inmunológicos. Previamente, nuestro grupo demostró que la infección aguda con la cepa virulenta RA regula negativamente la expresión de moléculas del CMHII en células presentadoras de antígeno (CPA) y altera la capacidad de células dendríticas (CD) esplénicas de estimular células T. Dado que las CD poseen un rol central en el inicio y desarrollo de la respuesta inmune, en el siguiente trabajo de tesis analizamos la capacidad del estadio de tripomastigote (Tp) de modular el estado de diferenciación y funcionalidad de CD derivadas de médula ósea in vitro. En dicho modelo, observamos que específicamente el estadio de Tp de T. cruzi fracasa a la hora de inducir activación de CD. Estas preservan la expresión basal de moléculas del CMHII y coestimulatorias, así como su capacidad endocítica. Asimismo, los Tp inducen secreción de TGF- β e incrementan la producción de interleuquina (IL)-10, observándose un aumento de la relación IL-10/IL-12p70 durante el tratamiento conjunto con LPS. Además, los Tp modulan la activación de CD inducida por LPS, regulando negativamente la expresión del CMHII y su capacidad de estimular linfoproliferación. La neutralización de IL-10 durante la diferenciación de las CD in vitro en presencia de Tp+LPS, revierte parcialmente la incapacidad de las CD de inducir alorespuesta durante una reacción linfocitaria mixta (RLM). Contrariamente, la neutralización simultánea de IL-10 y TGF-β durante la RLM no modifica la falta de alorespuesta observada. Ambas citoquinas, TGF-β e IL-10 son inmunomoduladoras y se encuentran asociadas a la inducción de tolerancia. Por lo tanto, estos resultados demuestran por primera vez que los Tp modulan la diferenciación de CD en presencia de LPS in vitro. Dichas CD presentan propiedades tolerogénicas (CDreg). Asimismo, Tp muertos por calor (Tpmc), pero no fijados con paraformaldehído o productos de excreción secreción parasitaria inducen CDreg. Las CD son responsables de modular la respuesta de células T, inclusive mediante la diferenciación de células T regulatorias (Tregs). Aquí se describe que tanto los Tp vivos como Tpmc alteran la capacidad de CD activadas con LPS de estimular linfocitos. En el cultivo de células T con CDreg se detectó producción de IL-10 y bajos niveles de IFN-γ. Asimismo, las CDreg alteran la respuesta ag-específica in vitro (OVA), utilizando células T CD4+ purificadas de ratones OT-II. Las células T CD4+ diferenciadas en presencia de CDreg (inducidas por Tp o Tpmc), muestran una menor expresión del marcador de activación temprana CD69 en comparación con las estimuladas con CD activadas con LPS. Además, estos CD4+ suprimen la alorespuesta en el marco de una RLM secundaria. Pese a no detectar diferencias respecto al número de Tregs CD4+CD25+FoxP3+, los resultados descriptos sugieren que las CD con propiedades tolerogénicas inducidas por T. cruzi, in vitro diferencian células T CD4+ potencialmente supresoras. La activación de la respuesta inmune depende del reconocimiento de señales de daño. El reconocimiento de patrones moleculares asociados a patógenos (PAMPs) por CD, juega un importante papel en el desarrollo de inmunidad. Numerosas moléculas de T. cruzi, capaces de activar CPA, han sido descriptas como ligandos de TLR2. Sin embargo, el modelo en CD se encuentra pobremente caracterizado. Las CDreg son inducidas por Tpmc, independientemente de infección. El fenotipo regulatorio se caracteriza por un estado particular de activación de CD: aumento en la activación de ERK 1/2 inducida por LPS y fosforilación de STAT3. Asimismo, los Tp no alteran la activación de p38, degradación de IκB-α o translocación a núcleo de NF-κB inducida por LPS. La vía de ERK es central en la inducción del fenotipo tolerogénico ya que su bloqueo regula negativamente la producción de IL-10 y restaura la capacidad de estimulación de las CD. La activación de NF-kB pero no la fosforilación de STAT3 está involucrada en la modulación de IL-10 por Tp. Un trabajo reciente demuestra que la señalización conjunta vía TLR2 y TLR4 induce sinergismo en la liberación de citoquinas anti-inflamatorias en CD murinas. El estímulo de TLR2 y TLR4 utilizando Pam3Cys o LPS y Tpmc en CD de ratones TLR2KO o mutantes para el TLR4, demuestra que los altos niveles de IL-10 son independientes del reconocimiento del parásito por TLR2 pero asociados a la señalización vía TLR4. En conclusión, estos resultados sugieren la importancia de la vía de ERK, la señalización por TLR4 y activación de NF-kB en la modulación de IL-10 inducida por T. cruzi y sugiere la existencia de interacciones parásito-CD aún no descriptas. La modulación de la diferenciación de CD por T. cruzi, puede explicarse como una estrategia de evasión desplegada por el parásito sobre una célula con potencial de desarrollar inmunidad con subsecuente control y erradicación de la infección. Sin embargo, muchos de los mecanismos inmunomoduladores que explican la persistencia parasitaria, favorecen el control de una respuesta proinflamatoria exacerbada, contrarrestándose el daño tisular concomitante. Futuros estudios permitirán descifrar el papel e impacto de los mecanismos inmunomoduladores en el balance entre tolerancia e inmunidad durante la infección experimental.A main feature of acute infection with Trypanosoma cruzi is the presence of immunological disorders. Previously, our group demonstrated that acute infection with the virulent RA strain downregulates the expression of MHCII on antigen presenting cells (APC) and impairs the T-cell stimulatory capacity of splenic dendritic cells (DC). Since DC have a main role in the initiation and subsequent development of immune responses, here we assess the ability of trypomastigotes (Tp) to modulate the differentiation stage and functionality of bone marrow-derived DC in vitro. In this model, we observe that specifically Tp stage of T. cruzi fails to activate DC, which preserved low expression of MHCII and costimulatory molecules as well as endocytic activity. We also describe that Tp induce TGF-β secretion by DC, and enhance the gap between interleukin (IL)-10 and IL-12 p70 production, showing a higher IL-10/IL-12p70 ratio upon LPS-treatment. In addition, we observe that Tp modulate full activation induced by LPS, thereby downregulating their MHCII surface expression and inhibiting DC capacity to stimulate lymphocyte proliferation. In vitro IL-10 neutralization during DC-differentiation process with Tp+LPS, partially reverts poor aloresponse during mixed lymphocyte reaction (MLR). In contrast, simultaneous neutralization of both IL-10 and/or TGF-β during MLR does not restore linfoproliferation. Since both TGF-β and IL-10 are immunosuppressive cytokines related to the immunoregulation and tolerance induction, our results suggest for the first time that Tp modulate LPS-treated DC differentiation in vitro. DC described, display tolerogenic properties (DCreg). In addition, Tp heat-killed (Tphk), but not fixed with paraformaldehyde or parasite excretion-secretion products induce the DC-differentiation profile described for live-Tp. DC modulate T cells responses also by inducing differentiation of regulatory T cells. In the present work we describe that live or heat-killed Tp affect T-cell stimulatory capacity of LPS-treated DC. T cells cultured with DCreg show impaired aloresponse, produce IL-10 and low levels of IFN-γ. DCreg are unable to induce ag-specific responses to OVA using CD4+ T cells from OT-II mice in vitro. Alogenic CD4+ T cells primed with DCreg (induced by live or Tphk) show reduce expression of the early-activation marker CD69 compared with the ones cultured with activated DC (LPS). In addition, CD4+ T cells primed with DCreg suppress aloresponse in the context of a secondary MLR. Eventhough, we fail to find CD4+CD25+FoxP3+ Tregs, these results suggest DC with tolerogenic properties induced by T. cruzi prime potentially regulatory CD4+ T cells. Activation of immune responses depends on recognition of danger signal. Recognition of pathogens associated molecular pattern (PAMPs) by DC plays a key role in the first steps related to development of immunity. Several T. cruzi molecules that stimulate APC were described as Toll-like receptor 2 (TLR2) ligands. However, DC models are poorly characterized. Here, we show that DCreg are induced by Tphk stimulation, ruling out infection as a key mechanism involved in Tp-DC modulation. Regulatory phenotype is characterized by a particular DC activation state, with increased LPS-induced activation of extracellular regulated kinase (ERK) 1/2 and phosphorylation of signal transducer and activator of transcription (STAT) 3. We also observe that Tp does not affect p38 activation, IκB-α degradation or NF-κB translocation to the nucleus induced by LPS. ERK has a central role in DC differentiation since its blockade down-regulates IL-10 production and restores DC stimulatory capacity. NF-kB activation but not STAT3 phosphorylation is associated to IL-10 modulation by Tp. A recent work shows that signaling via TLR4 and TLR2 induces a synergism in anti-inflammatory cytokine production in murine DC. Upon TLR2 and TLR4 stimulation using Pam3Cys or LPS and Tphk in DC from TLR2 knock out (KO) or TLR4-mutant mice, we show that high levels of IL-10 are independent of TLR2 but associated to TLR4 signallization. In conclusion, all these findings demonstrate a key role of ERK and TLR4 in association to NF-kB in IL-10 modulation induced by T. cruzi and suggest that this regulatory effect involves parasite-DC interactions not described yet. Modulation of DC differentiation program by T. cruzi might be an evasion mechanism displayed by the parasite with immunosuppressive effects and consequences in the eradication of the parasite. However, immunoregulatory mechanisms in general associated with parasite persistence, may also control pro- inflammatory responses and counteract host tissue damage. Future goals will undercover the role and impact of immunomodulation in the balance between tolerance and immunity during the experimental infection in vivoFil:Poncini, Carolina Verónica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Trypanosoma cruzi Induces Regulatory Dendritic Cells In Vitro▿

A main feature of acute infection with Trypanosoma cruzi is the presence of immunological disorders. A previous study demonstrated that acute infection with the virulent RA strain downregulates the expression of major histocompatibility complex class II (MHC-II) on antigen-presenting cells and impairs the T-cell stimulatory capacity of splenic dendritic cells (DC). In the present work, we assessed the ability of trypomastigotes (Tp) to modulate the differentiation stage and functionality of bone marrow-derived DC in vitro. We observed that the Tp stage of T. cruzi failed to activate DC, which preserved their low expression of MHC-II and costimulatory molecules, as well as their endocytic activity. We also show that Tp induced transforming growth factor β (TGF-β) secretion by DC and enhanced the gap between interleukin-10 (IL-10) and IL-12p70 production, showing a higher IL-10/IL-12p70 ratio upon lipopolysaccharide (LPS) treatment. In addition, we observed that Tp prevented DC full activation induced by LPS, thereby downregulating their MHC-II surface expression and inhibiting their capacity to stimulate lymphocyte proliferation. In vitro IL-10 neutralization during the differentiation process of DC with Tp+LPS showed a reversion of their inhibitory effect during mixed lymphocyte reaction. In contrast, only simultaneous neutralization of IL-10 and TGF-β, after DC differentiation, was involved in the partial restitution of lymphocyte proliferation. Since both TGF-β and IL-10 are immunosuppressive cytokines essential in the modulation of the immune response and important in the induction of tolerance, our results suggest for the first time that Tp are responsible for the generation of regulatory DC in vitro

Lipids from attenuated and virulent Babesia bovis strains induce differential TLR2-mediated macrophage activation

Babesia bovis is an intraerythrocytic apicomplexan protozoa of cattle that causes an acute infection with parasite persistence. Babesiosis limitation depends on macrophages, essential effector cells of the host innate defense, which generate inflammatory cytokines and nitric oxide. Herein, we report quantitative differences in the lipid composition of merozoites from two B. bovis strains with polar behaviour: attenuated R1A and virulent S2P. Accordingly, we observed a distinct inflammatory response induced by the total lipids of R1A (L(A)) and S2P (L(V)) in murine peritoneal macrophages. L(A) and particularly its fractions phosphatidic acid and phosphatidylserine+phosphatidylinositol (PS+PI), produced a strong activation of these cells with lipid body formation, cyclooxygenase-2 expression and pro-inflammatory TNFalpha, IL-6 and KC secretion. Although L(V) did not activate these cells, the corresponding PS+PI fraction induced TNFalpha, IL-6 and KC release. Therefore, these facts might be suggesting the presence of an inhibitor in L(V). Furthermore, the employment of wild type and toll like receptor 2 knockout (TLR2KO) mice allowed us to demonstrate that macrophage activation by the stimulating lipid fractions was mediated through TLR2. Interestingly, only L(A) activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Inhibitory studies employing UO126, indicated that the ERK pathway was required for TNFalpha, IL-6 and KC release. In conclusion, the absence of inflammatory response observed with the lipids of S2P virulent strain could constitute an evasion mechanism of the innate immune response enabling parasite establishment in the host.Fil: Gimenez, G.. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; ArgentinaFil: Magalhães, K. G.. Fundación Oswaldo Cruz; BrasilFil: Belaunzarán, María Laura. 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. Departamento de Microbiología; ArgentinaFil: Poncini, Carolina Verónica. 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. Departamento de Microbiología; ArgentinaFil: Lammel, Estela María. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; 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: Gonzalez Cappa, S. M.. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; ArgentinaFil: Bozza, P. T.. Fundación Oswaldo Cruz; BrasilFil: Isola, E .L .D.. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentin