PARTICIPACIÓN DEL RECONOCIMIENTO INMUNE INNATO EN LA RESPUESTA DE CARDIOMIOCITOS DURANTE LA INFECCIÓN EXPERIMENTAL CON TRYPANOSOMA CRUZI. Characterization of the cardiac innate immune response during Trypanosoma cruzi experimental infection.

La enfermedad de Chagas, causada por Trypanosoma cruzi, constituye la principal miocarditis infecciosa a nivel mundial. Crecientes evidencias revelan que la respuesta inmune innata tendría un rol determinante en la fisiopatología de las enfermedades cardiovasculares. La inmunidad innata es la primera línea de defensa, no específica, preprogramada para combatir agentes infecciosos. Este sistema censa la presencia de antígenos extraños a través de los receptores tipo toll (TLR) produciendo citoquinas y activando mecanismos microbicidas. Sin embargo, los TLRs también se hayan distribuidos en las células parenquimales no inmunes, jugando un importante rol tanto en la defensa como en la homeostasis de cada tejido. Durante la etapa aguda de la infección, el T. cruzi invade y se replica dentro de una amplia variedad de células y tejidos. Pero posteriormente, los parásitos son efectivamente eliminados de la mayoría de los tejidos persistiendo durante toda la vida en las células del músculo cardíaco y esquelético de los pacientes infectados. Debido a que el mantenimiento de la célula cardíaca infectada es crítica para la patogénesis de la enfermedad, los mecanismos que participan en la sobrevida de los cardiomiocitos están siendo foco de nuestro estudio. Hemos demostrado, que la infección ejerce efectos antiapoptóticos sobre células cardíacas aisladas. Nuestra hipótesis es que la inmunidad innata cardíaca estaría involucrada en el mantenimiento de la sobrevida de los miocitos así como en la defensa contra el parásito. Objetivo general: determinar la participación de la respuesta inmune innata cardíaca en el desarrollo de la enfermedad de Chagas experimental murina. Objetivos específicos: 1) Analizar el compromiso de TLRs en la respuesta anti-apoptótica y de autofagia de cardiomiocitos aislados de ratones salvajes y de ratones deficientes en TLR4, TLR2 y en MyD88, molécula adaptadora de la señalización por TLRs, sometidos a la infección con el parásito. 2) Determinar la importancia de la actividad cisteín proteasa parasitaria en el grado de infectividad y la sobrevida de cultivos primarios de ratones salvajes infectados con parásitos transgénicos que poseen disminuída o nula actividad cisteín proteasa. 3) Establecer la cinética de expresión de TLR2/TLR6, TLR4 y TLR9, factores antiapoptóticos (Bcl-2, Bcl-xL, etc.), daño cardíaco y la carga parasitaria en el tejido cardíaco de ratones infectados salvajes y/o deficientes antes mencionados. Materiales y Métodos: Los animales serán infectados i.p. con 5x103 parásitos y se determinará la cinética de expresión de los mediadores mencionados por western blot e inmunofluorescencia, la carga parasitaria será determinada por qRT-PCR. Como controles se procesarán animales inyectados con solución salina. En cultivos primarios de cardiomiocitos de ratones neonatos salvajes y deficientes infectados se estudiará la carga parasitaria, la activación de los mecanismos microbicidas (producción de óxido nítrico, metabolitos reactivos del oxígeno y del nitrógeno, ciclooxigenasa, etc.), producción de citoquinas y expresión de moléculas anti-apoptóticas (Bcl-2, Bcl-xL, Bax, etc.). Se explorará la tasa de apoptosis en cultivos deprivados de suero. La autofagia se analizará por microscopia electrónica. Cultivos controles serán mantenidos en medio o tratados con ligandos de los diferentes TLRs. Resultados preliminares sugieren que tanto TLR2 como Bcl-2 se incrementan en tejido cardíaco infectado. Esto nos lleva a profundizar en los mecanismos observados en cultivos y estudiarlos en un modelo in vivo, analizando la posible importancia que tiene la inmunidad innata cardíaca en el control del establecimiento de la infección. La comprensión de los mecanismos que mantienen la sobrevida de los cardiomiocitos y su respuesta a la infección es importante ya que el conocimiento de las bases moleculares es fundamental para el desarrollo de nuevos agentes quimioterapéuticos. Chagas disease is endemic in Central and South America and causes the most common myocarditis worldwide. We have previously reported that the cardiotrophic parasite Trypanosoma cruzi, its etiological agent, protects cardiomyocytes against apoptosis induced by growth factor deprivation activating the PI3K/Akt and MEK1/ERK signaling pathways. Recent studies have shown that local innate immunity plays a key role in initiating and coordinating homeostatic as well as defense responses in the heart. One of the mechanisms by which the innate immune system senses the presence of foreign antigens is through TLRs. The stimulation of these receptors leads to the activation and nuclear translocation of NF-kB transcription factor and the production of cytokines. Proinflammatory cytokines, in turn, appear to play a central role in the orchestration and timing of the intrinsic cardiac stress response providing, under different situations, instantaneous anti-apoptotic cytoprotective signals, which allow tissue repair and/or remodeling. The aim of the present project is to study the cardiomyocyte innate immune responses to T. cruzi infection and its role in target cell protection from apoptosis. Specific objectives: 1) Study the mechanism triggered by TLR in the anti-apoptotic response and parasite load of infected cardiomyocyte primary cultures from wild type and mice deficient in TLR2, TLR4 or MyD88. 2) Determine the effect of parasite cisteín protease activity on primary cultures from wild type mice. 3) Determine the TLR signaling-involvement in parasite load and survival indicators in deficient mice. Preliminary results showed us that cardiac-TLR2 may be involved in the anti-apoptotic effect elicited by the parasite and prompted us to establish the mechanisms triggered by the innate immunity that mediate parasite persistence within the host cell.

Estudi de la resposta immune enfront de Neospora caninum

S’investiga la resposta immune que té lloc en la infecció experimental de les vedelles gestants amb Neospora caninum a les 3, 6 i 9 setmanes postinfecció, amb especial interès en els teixits placentaris. Donada la importancia que tenen les citoquines en la resposta immune a la infecció, s’ha posat a punt les tècniques de RT-PCR a temps real per a l’estudi de l’expressió génica de citoquines del boví. En una fase posterior s’ha estudiat la dinàmica dels anticossos durant l’experiment, la transmissió transplacentària mitjançant l’anàlisi de la presència de DNA parasitari en teixits, la relació entre la producció de IFN-gamma en fluids fetals i, els avortaments i la trasmissió transplacentària i, finalment, el patró de citoquines, amb èmfasi en la interfase materno-fetal.

Chagas' disease: selective affinity and cytotoxicity of Trypanosoma cruzi-immune lymphocytes to parasympathetic ganglion cells

The megaesophagus and megacolon endemic in South America are related , to Chagas' disease. These mega conditions are found in patients with chronic Chagas's infection, when the parasite is not demonstrable in the lesions. These are characterized by depopulation of parasympathetic ganglion cells, dilation and hypertrophy of the viscera. In the experiments described here we deminstrate a selective affinity and adherence of Trypanosoma cruzi-immune lymphocytes to myenteric, parasympathetic ganglion cells, leading to neuronolysis. None of these features are observed when non-immune lymphocytes from control rabbits are used, or when the immune lymphocytes are allowed to react with CNS neurons. This demonstration is an indication of the high degree of specificity of the destruction of parasympathetic neurons in Chagas' disease. We postulate that the T. cruzi-immune lymphocyte rejection of parasympathetic neurons, but not of CNS neurons, might be related to recognition of a cross-reacting antigenic determinant secreted only by the target neurons. In favor of this interpretation is the observation of lymphocytic infiltrates and parasympathetic ganglion cell destruction in chronic Chagas' infection in the absence of encephalitis.

Cross-suppression of specific immune responses after oral tolerance

Adult normal inbred mice rendered tolerant to OVA by previous oral exposure do not respond to intraperitonela immunization with DNP-OVA in adjuvant. These tolerant mice also form less DNP-specific antibodies to DNP-KLH when immunized with mixtures of DNP-KLH and DNP-OVA, or less HGG-specific antibodies when immunized with cross-linked conjugates of OVA and HGG. These same procedures increased DNP-specific or HGG-specific responses in non-tolerant control mice. The cross-supperssion was ineffective, however, to inhibit already ongoing antibody responses.

Contribution to the study of immune hemolysis by toad complement

EA (sheep erythrocytes carrying rabbit antibody) are lysed by toad complement under optimal conditions which include a low concentration of cells (1.54 x 10*8/ml), a low temperature of incubation (30°C) and the same amounts of Ca++ and Mg++ as required for the titration of guinea-pig complement. Kinetic studies of the role of cations mentioned above in immune lysis by toad C have disclosed a fundamental difference as compared to guinea-pig C. In a limited complement system, the lysis by amphibian C is completely blocked by EDTA, even when the chelating agent is added as late as 15 minutes after zero-time. Inhibition by EGTA is only partial and the findings suggest that Mg++ is required not only at the beginning, but also at late stages of the lytic process. It has been speculated that the activation of amphibian complement proceeds mainly by the alternative pathway.

Functional analysis of the murine T lymphocyte immune response to a protozoan parasite, Leishmania tropica

The results presented in this review summarize a seirs of experiments designed to characterize the murine T cell imune response to the protozoan parasite Leishmania tropica. Enriched T cell populations and T cell clones specific for L. tropica antigens were derived from lymph nodes of primed mice and maintained in continous culture in vitro. These T lymphocytes were shown (A) to express the Lyt 1+ 3- cell surface phenotype, (B) to proliferate specifically in vitro in response to parasite antigens, together with a source of irradiated syngeneic macrophages, (C) to transfer antigen-specific delayed-type hypersensitivity (DTH) responses to normal syngeneic mice, (D) to induce specific activation of parasitized macrophages in vitro resulting in the destruction of intracellular parasites, (E) to provide specific helper activity for antibody responses in vitro in a hapten-carrier system. Protection studies using these defiened T cell populations should allow the characterization of parasite antigen(s) implicated in the induction of cellular immune responses beneficial for the host.

Loss of Cutaneous TSLP-Dependent Immune Responses Skews the Balance of Inflammation from Tumor Protective to Tumor Promoting.

Inflammation can promote or inhibit cancer progression. In this study we have addressed the role of the proinflammatory cytokine thymic stromal lymphopoietin (TSLP) during skin carcinogenesis. Using conditional loss- and gain-of-function mouse models for Notch and Wnt signaling, respectively, we demonstrate that TSLP-mediated inflammation protects against cutaneous carcinogenesis by acting directly on CD4 and CD8 T cells. Genetic ablation of TSLP receptor (TSLPR) perturbs T-cell-mediated protection and results in the accumulation of CD11b(+)Gr1(+) myeloid cells. These promote tumor growth by secreting Wnt ligands and augmenting β-catenin signaling in the neighboring epithelium. Epithelial specific ablation of β-catenin prevents both carcinogenesis and the accumulation of CD11b(+)Gr1(+) myeloid cells, suggesting tumor cells initiate a feed-forward loop that induces protumorigenic inflammation.

Intraseasonal variation in immune defence, body mass and hematocrit in adult house martins Delichon urbica

The intensity of parasite infections often increases during the reproductive season of the host as a result of parasite reproduction, increased parasite transmission and increased host susceptibility. We report within-individual variation in immune parameters, hematocrit and body mass in adult house martins Delichon urbica rearing nestlings in nests experimentally infested with house martin bugs Oeciacus hirundinis and birds rearing nestlings in initially parasite-free nests. From first to second broods body mass and hematocrit of breeding adult house martins decreased. In contrast leucocytes and immunoglobulins became more abundant. When their nests were infested with ectoparasites adults lost more weight compared with birds raising nestlings in nests treated with pyrethrin, whereas the decrease in hematocrit was more pronounced during infection with blood parasites. Neither experimental infestation with house martin bugs nor blood parasites had a significant effect on the amount of immune defences.

Neuroendocrine, metabolic, and immune functions during the acute phase response of inflammatory stress in monosodium L-glutamate-damaged, hyperadipose male rat.

In rats, neonatal treatment with monosodium L-glutamate (MSG) induces several metabolic and neuroendocrine abnormalities, which result in hyperadiposity. No data exist, however, regarding neuroendocrine, immune and metabolic responses to acute endotoxemia in the MSG-damaged rat. We studied the consequences of MSG treatment during the acute phase response of inflammatory stress. Neonatal male rats were treated with MSG or vehicle (controls, CTR) and studied at age 90 days. Pituitary, adrenal, adipo-insular axis, immune, metabolic and gonadal functions were explored before and up to 5 h after single sub-lethal i.p. injection of bacterial lipopolysaccharide (LPS; 150 microg/kg). Our results showed that, during the acute phase response of inflammatory stress in MSG rats: (1) the corticotrope-adrenal, leptin, insulin and triglyceride responses were higher than in CTR rats, (2) pro-inflammatory (TNFalpha) cytokine response was impaired and anti-inflammatory (IL-10) cytokine response was normal, and (3) changes in peripheral estradiol and testosterone levels after LPS varied as in CTR rats. These data indicate that metabolic and neroendocrine-immune functions are altered in MSG-damaged rats. Our study also suggests that the enhanced corticotrope-corticoadrenal activity in MSG animals could be responsible, at least in part, for the immune and metabolic derangements characterizing hypothalamic obesity.

Covariation between colony social structure and immune defences of workers in the ant Formica selysi

Several ant species vary in the number of queens per colony, yet the causes and consequences of this variation remain poorly understood. In previous experiments, we found that Formica selysi workers originating from multiple-queen (=polygyne) colonies had a lower resistance to a fungal pathogen than workers originating from single-queen (=monogyne) colonies. In contrast, group diversity improved disease resistance in experimental colonies. This discrepancy between field and experimental colonies suggested that variation in social structure in the field had antagonistic effects on worker resistance, possibly through a down-regulation of the immune system balancing the positive effect of genetic diversity. Here, we examined if workers originating from field colonies with alternative social structure differed in three major components of their immune system. We found that workers from polygyne colonies had a lower bacterial growth inhibitory activity than workers from monogyne colonies. In contrast, workers from the two types of colonies did not differ significantly in bacterial cell wall lytic activity and prophenoloxidase activity. Overall, the presence of multiple queens in a colony correlated with a slight reduction in one inducible component of the immune system of individual workers. This reduced level of immune defence might explain the lower resistance of workers originating from polygyne colonies despite the positive effect of genetic diversity. More generally, these results indicate that social changes at the group level can modulate individual immune defences.