Felinized murine intestinal organoids for gaining insight into sexual reproduction of Toxoplasma gondii

The apicomplexan parasite Toxoplasma gondii is the causative agent of Toxoplasmosis, a zoonotic disease affecting one-third of the human population which can cause severe fetal damage by vertical transmission in pregnancy. Toxoplasmosis has great impact in reproductive outcomes of productive species and thus economic losses worldwide. The life cycle of T. gondii encompasses sexual and asexual phases. The asexual cycle can occur in any warm-blooded animal while the sexual stage is restricted to felids. In the feline enterocytes, bradyzoites turn into merozoites, initiating sexual replication that will end in the formation of a zygote. Zygotes are key to the generation of diversity, as they allow the occurrence of genetic admixing and the generation of oocyst that will be disseminated in the environment with the feline feces. Until recently, limitations in the availability of appropriate experimental models had hindered the study of sexual stages despite its clear biological relevance. In the last year, the molecular basis involved in the species specificity of the sexual parasitic forms was identified: an excess of linoleic acid given by the lack of delta-6-desaturase activity in the felid´s intestine. Thus, mimicking these conditions in a murine intestine allowed T. gondii to sexually develop in a mouse model, providing the opportunity to answer biological questions relevant to T. gondii reproduction without the need of using feline animal models. This work aimed to set up culturing systems based on “felinized” murine intestinal organoids in order to trigger T. gondii’s differentiation into sexual stages in vitro. For this purpose, murine intestinal organoids generated from isolated intestinal stem cells and maintained in a 3D system inside a matrix, or trypsinized and seeded as a monolayer, were incubated in the presence of 20 µM delta-6-desaturase inhibitor and 200 µM linoleic acid. The cytotoxicity of felinizing compounds in 2D and 3D cultures was assessed showing no cytotoxicity for 5 days of culture. Optimization of the infection assays was performed by incubating intestinal organoids with bradyzoites, at two multiplicities of infection (1:1 and 1:10). The presence of the parasite was evaluated after 5 days of culture by immunofluorescence. Kinetic studies of the sexual differentiation of T. gondii were carried out and the evaluation of parasite switching efficiency to sexual stages is ongoing, both by immunofluorescence and qRT-PCR. We put forward in vitro felinized intestinal organoids as a valuable tool for answering biological questions relevant to persistence and dissemination of T. gondii.Agencia Nacional de Investigación e Innovació

Exploring Toxoplasma gondii´s Biology within the Intestinal Epithelium: intestinal-derived models to unravel sexual differentiation

A variety of intestinal-derived culture systems have been developed to mimic in vivo cell behavior and organization, incorporating different tissue and microenvironmental elements. Great insight into the biology of the causative agent of toxoplasmosis, Toxoplasma gondii, has been attained by using diverse in vitro cellular models. Nonetheless, there are still processes key to its transmission and persistence which remain to be elucidated, such as the mechanisms underlying its systemic dissemination and sexual differentiation both of which occur at the intestinal level. Because this event occurs in a complex and specific cellular environment (the intestine upon ingestion of infective forms, and the feline intestine, respectively), traditional reductionist in vitro cellular models fail to recreate conditions resembling in vivo physiology. The development of new biomaterials and the advances in cell culture knowledge have opened the door to a next generation of more physiologically relevant cellular models. Among them, organoids have become a valuable tool for unmasking the underlying mechanism involved in T. gondii sexual differentiation. Murine-derived intestinal organoids mimicking the biochemistry of the feline intestine have allowed the generation of pre-sexual and sexual stages of T. gondii for the first time in vitro, opening a window of opportunity to tackling these stages by “felinizing” a wide variety of animal cell cultures. Here, we reviewed intestinal in vitro and ex vivo models and discussed their strengths and limitations in the context of a quest for faithful models to in vitro emulate the biology of the enteric stages of T. gondii.Pasteur NetworkFondo para la Convergencia Estructural del MERCOSURAgencia Nacional de Investigación e InnovaciónPrograma de Desarrollo de las Ciencias Básicas (PEDEClBA

Murine colon organoids as a novel model to study Trypanosoma cruzi infection and interactions with the intestinal epithelium

Chagas disease (CD) is a life-threatening illness caused by the parasite Trypanosoma cruzi (T. cruzi). With around seven million people infected worldwide and over 50,000 deaths per year, CD is a major public health issue in Latin America. The main route of transmission to humans is through a triatomine bug (vector-borne), but congenital and oral transmission have also been reported. The acute phase of CD presents mild symptoms but may develop into a long-lasting chronic illness, characterized by severely impaired cardiac, digestive, and neurological functions. The intestinal tissue appears to have a key role during oral transmission and chronic infection of CD. In this immune-privileged reservoir, dormant/quiescent parasites have been suggested to contribute to disease persistence, infection relapse, and treatment failure. However, the interaction between the intestinal epithelium and T. cruzi has not been examined in depth, in part, due to the lack of in vitro models that approximate to the biological and structural complexity of this tissue. Therefore, to understand the role played by the intestinal tissue during transmission and chronic infection, physiological models resembling the organ complexity are needed. Here we addressed this issue by establishing and characterizing adult stem cell-derived colonoid infection models that are clinically relevant for CD. 3D and 2D systems of murine intestinal organoids infected with T. cruzi Dm28c (a highly virulent strain associated with oral outbreaks) were analyzed at different time points by confocal microscopy. T. cruzi was able to invade and replicate in intestinal epithelial primary cells grown as intact organoids (3D) and monolayers (2D). The permissiveness to pathogen infection differed markedly between organoids and cell lines (primate and intestinal human cell lines). So far, this represents the first evidence of the potential that these cellular systems offer for the study of host-pathogen interactions and the discovery of effective anti-chagasic drugs.Agencia Nacional de Investigación e InnovaciónPasteur NetworkFOCEM (MERCOSUR Structural Convergence Fund

Modeling host-parasite interaction in chagas disease with murine intestinal organoids

Chagas disease (CD) is a potentially life-threatening illness caused by the parasite Trypanosoma cruzi (T. cruzi). With around seven million people infected worldwide and over 10,000 deaths per year, CD is a major public health issue in Latin America. The main route of transmission to humans is through a triatomine bug (vector-borne) and, to a minor extent, by blood transfusion, organ transplantation, laboratory accidents, congenitally and orally (food-borne). The acute phase of CD presents mild symptoms. If left untreated, it develops into a long-lasting chronic illness, characterized by severely impaired cardiac, digestive, and neurological functions. The intestinal tissue appears to have a key role during oral transmission and chronic infection of CD. In these immune-privileged reservoirs, dormant/quiescent parasites have been suggested to contribute to disease persistence, infection relapse, and treatment failure. However, the interaction between the intestinal epithelium and T. cruzi has not been examined in depth, in part, due to the lack of in vitro models resembling the biological and structural complexity of this organ. Therefore, to understand the pathophysiological role played by the intestinal tissue during transmission and chronic infection, we evaluated the progression of T. cruzi infection of murine colon organoids. In order to model CD, 3D and 2D systems of murine intestinal organoids were infected with T. cruzi Dm28c, a strain that has been associated with high virulence and oral outbreaks. At different time points, the presence and load of parasites in the organoids, as well as the host cell morphology were evaluated by confocal microscopy, and compared to those obtained with a classical infection model (Vero cells). We show that the parasite invades and replicates in intestinal epithelial primary cells grown as intact organoids (3D) and monolayers (2D). The permissiveness to pathogen infection differed markedly between the primary and the tumoral (Vero) cells. So far, this represents the first evidence of the potential of these nearly physiological cellular systems to study host-pathogen interaction for CD and/or for the future evaluation of anti-chagasic drugs.Agencia Nacional de Investigación e Innovación (ANII)FOCEM (MERCOSUR Structural Convergence Fund

Jejunum-derived NF-κB reporter organoids as 3D models for the study of TNF-alpha-induced inflammation

Inflammation is an important process for epithelial barrier protection but when uncontrolled, it can also lead to tissue damage. The nuclear factor-kappa light chain enhancer of activated B cells (NF-κB) signaling pathway is particularly relevant in the intestine, as it seems to play a dual role. Whereas NF-κB protects intestinal epithelium against various noxious stimuli, the same pathway mediates intestinal inflammatory diseases by inducing pro-inflammatory gene expression. The availability of appropriate in vitro models of the intestinal epithelium is crucial for further understanding the contribution of NF-κB in physiological and pathological processes and advancing in the development of drugs and therapies against gut diseases. Here we established, characterized, and validated three-dimensional cultures of intestinal organoids obtained from biopsies of NF-κB-RE-Luc mice. The NF-κB-RE-Luc intestinal organoids derived from different intestine regions recreated the cellular composition of the tissue and showed a reporter responsiveness similar to the in vivo murine model. When stimulated with TNF-α, jejunum-derived NF-κB-RE-Luc-reporter organoids, provided a useful model to evaluate the anti-inflammatory effects of natural and synthetic compounds. These reporter organoids are valuable tools to explore the epithelial TNF-α-induced NF-κB contribution in the small intestine, being a reliable alternative method while helping to reduce the use of laboratory animals for experimentation.Agencia Nacional de Investigación e InnovaciónFOCEM (MERCOSUR Structural Convergence Fund

Potencial de los organoides intestinales murinos para el estudio de la enfermedad de Chagas

Chagas disease (CD) is a potentially life-threatening illness caused by the parasite Trypanosoma cruzi (T. cruzi). With around seven million people infected worldwide and over 10,000 deaths per year, CD is a major public health issue in Latin America. The main route of transmission to humans is through a triatomine bug (vector-borne) and, to a minor extent, by blood transfusion, organ transplantation, laboratory accidents, congenitally and orally (food- borne). The acute phase of CD presents mild symptoms. If left untreated, it develops into a long-lasting chronic illness, characterized by severely impaired cardiac, digestive, and neurological functions. The intestinal tissue appears to have a key role during oral transmission and chronic infection of CD. In these immune-privileged reservoirs, dormant/quiescent parasites have been suggested to contribute to disease persistence, infection relapse, and treatment failure. However, the interaction between the intestinal epithelium and T. cruzi has not been examined in depth, in part, due to the lack of in vitro models resembling the biological and structural complexity of this organ. Therefore, to understand the pathophysiological role played by the intestinal tissue during transmission and chronic infection, we evaluated the progression of T. cruzi infection of murine colon organoids. In order to model CD, 3D and 2D systems of murine intestinal organoids were infected with T. cruzi Dm28c, a strain that has been associated with high virulence and oral outbreaks. At different time points, the presence and load of parasites in the organoids, as well as the host cell morphology were evaluated by confocal microscopy, and compared to those obtained with a classical infection model (Vero cells). We show that the parasite invades and replicates in intestinal epithelial primary cells grown as intact organoids (3D) and monolayers (2D). The permissiveness to pathogen infection differed markedly between the primary and the tumoral (Vero) cells. So far, this represents the first evidence of the potential of these nearly physiological cellular systems to study host-pathogen interaction for CD and/or for the future evaluation of anti-chagasic drugs.Agencia Nacional de Investigación e Innovación (ANII

Characterization of Oct4-GFP transgenic mice as a model to study the effect of environmental estrogens on the maturation of male germ cells by using flow cytometry

Oct4 is involved in regulation of pluripotency during normal development and is down-regulated during formation of postnatal reservoir of germ cells. We propose thatOct4/GFP transgenic mouse, which mimics the endogenous expression pattern of Oct4, could be used as a mammalian model to study the effects of environmental estrogens on the development of male germ cells. Oct4/GFP maturation profile was assessed during postnatal days -PND- 3, 5, 7, 10, 14 and 80, using flow cytometry. Then, we exposed pregnant mothers to 17a-ethinylestradiol (EE2) from day post coitum (dpc) 5 to PND7. Percentage of Oct4/GFP-expressing cells and levels of expression of Oct4/GPF were increased in PND7 after EE2 exposure. These observations were confirmed by analysis of GFP and endogenous Oct4 protein in the seminiferous tubules and by a reduction in epididymal sperm count in adult mice. We introduced Oct4/ GFP mouse together with flow cytometry as a tool to evaluate changes in male germ cells development.Agencia Nacional de Investigación e InnovaciónFONCyT (Fondo para la InvestigaciónCientífica y Tecnológica de la Agencia Nacional de PromociónCientífica y Tecnológica, ANPCyT, Argentina)FOCEM (MERCOSUR Structural Convergence Fund), COF 03/11CONICET (becas doctoral y postdoctoral