The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections. However, current in vitro models do not allow long-term culture of these cysts to maturity. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts that are orally infectious to mice and tolerate exposure to a range of antibiotics and temperature stresses. Metabolomic characterization of purified cysts reveals global changes that comprise increased levels of amino acids and decreased abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate. Direct access to persistent stages of T. gondii under defined cell culture conditions will be essential for the dissection of functionally important host-parasite interactions and drug evasion mechanisms. It will also facilitate the identification of new strategies for therapeutic intervention.

Mounting evidence argues for the significant impact of sex in numerous cardiac pathologies, including myocarditis. Macrophage polarization and activation of cardiac fibroblasts play a key role in myocardial inflammation and remodeling. However, the role of sex in these processes is still poorly understood. In this study, we investigated sex-specific alterations in the polarization of murine bone marrow-derived macrophages (BMMs) and the polarization-related changes in fibroblast activation. Cultured male and female murine BMMs from C57/BL6J mice were polarized into M1 (LPS) and M2 (IL-4/IL-13) macrophages. Furthermore, male and female cardiac fibroblasts from C57/BL6J mice were activated with TNF-α, TGF-β, or conditioned medium from M1 BMMs. We found a significant overexpression of M1 markers (c-fos, NFκB, TNF-α, and IL-1β) and M2 markers (MCP-1 and YM1) in male but not female activated macrophages. In addition, the ROS levels were higher in M1 male BMMs, indicating a stronger polarization. Similarly, the pro-fibrotic markers TGF-β and IL-1β were expressed in activated cardiac male fibroblasts at a significantly higher level than in female fibroblasts. In conclusion, the present study provides strong evidence for the male-specific polarization of BMMs and activation of cardiac fibroblasts in an inflammatory environment. The data show an increased inflammatory response and tissue remodeling in male mice.

SummaryThe apicomplexan parasite Toxoplasma gondii causes chronic and drug-tolerant infections, yet current models do not permit metabolomic characterization of these persisting tissue cysts. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts that enabled direct measurements of their metabolome. The cysts are functionally mature and tolerate exposure to a range of antibiotics and to extended temperature stresses and are orally infectious to mice. Metabolomic characterization of purified cysts reveals global changes that comprise systematically increased levels of amino acids and decrease abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. Consequently, pharmacological modulation of the TCA cycle in T. gondii bradyzoites reveals that this pathway is rendered dispensable during parasite stage conversion. Direct access to persisting parasite stages will be essential for the dissection of functionally important host-parasite interactions and drug evasion mechanisms and also help to identify new strategies for therapeutic intervention.Highlights–Toxoplasma gondii forms mature tissue cysts in immortalized human myotubes–In vitro cysts of T. gondii develop drug tolerance and temperature stress resistance–Untargeted metabolomic characterization of tissue cysts reveals a distinct metabolome–The mitochondrial tricarboxylic acid cycle is dispensable in T. gondii bradyzoites