Characterization of L. infantum infection in hepatocyte organoid immune cell co-cultures

Abstract

Visceral leishmaniasis is a neglected tropical disease caused by a systemic infection with protozoan parasites of the genus Leishmania. Throughout the course of the disease, tissue-resident macrophages in the liver (Kupffer cells) are infected. This initiates a pivotal immune response essential for parasite elimination. To date, little is known about the degree to which functional liver cells, such as hepatocytes, are directly impacted by the infection and contribute to the development of this immune response. Furthermore, the investigation of underlying immune mechanisms is impeded by the absence of appropriate model systems. In this study, an in vitro co-cultivation system was established, that integrates murine hepatocyte organoids with monocytes or macrophages, to simulate hepatic infection with Leishmania infantum parasites. The initial part of this study demonstrated the successful generation of three-dimensional hepatocyte organoids derived from both human and murine primary hepatocytes. The functionality as well as the proliferative capacity of the cultured organoids was proven. Additionally, it was illustrated that under appropriate culture conditions, L. infantum parasites could infiltrate the generated hepatocyte organoids. The resulting immune response of the organoids was comprehensively investigated through cytokine and transcriptome analyses. Overall, both methodologies revealed only minimal responsiveness of the hepatocyte organoids to the infection. Moreover, a total of five distinct systems for co-cultivating hepatocyte organoids with either monocytes or macrophages were applied, distinguished primarily by the utilization of a hydrogel that mimics the extracellular matrix. Infection with L. infantum parasites within these co-cultures was represented either through prior infection of murine organoids, following the established methodology, or by infection of murine macrophages. To assess the efficacy of these systems, the dynamics in cytokine production in comparison to the murine model were analyzed via ELISA and Multiplex Cytokine assay. Co-cultivation of murine monocytes with L. infantum-infected hepatocyte organoids in suspension resulted in an infection-specific induction of cytokines, including CCL3, CCL2, TNF, IFN-g and IL-10, aligning with trends observed in the murine experimental model of VL. Furthermore, it was observed that the induction of specific cytokines, such as CCL3 and TNF, was exclusive to co-cultures including both hepatocyte organoids and monocytes. Additionally, utilizing qPCR, infection-specific differences in the transcript levels of Nos2 and Arg1 were discerned, indicating upregulation of Nos2 and downregulation of Arg1 following infection, consistent with mechanisms typically associated with the elimination of parasites. These findings provide insights into the role of hepatocytes in modulating liver microenvironment conducive to the eradication of Leishmania parasites. The replication of infection-specific cytokine dynamics observed in the animal model within the in vitro co-cultivation system of hepatocyte organoids and monocytes demonstrated in this study, underscores the potential of this methodology to advance the reduction of animal experiments in line with the 3R principle (Reduce, Refine, Replace). Furthermore, these established techniques offer a platform for future investigations utilizing human specimens, thereby enriching the current understanding of immune mechanisms during visceral leishmaniasis in humans.