Iron is a trace metal element that is essential for the survival of cells and parasites. The role of iron in cerebral toxoplasmosis (CT) is still unclear. Deferiprone (DFP) is the orally active iron chelator that binds iron in a molar ratio of 3:1 (ligand:iron) and promotes urinary iron excretion to remove excess iron from the body. The aims of this experiment were to observe the alterations in iron in brains withToxoplasma gondii(T.gondii) acute infections and to investigate the mechanism of ferroptosis in CT using DFP. We established a cerebral toxoplasmosis modelin vivousing TgCtwh3, the dominant strains of which are prevalent in China, and treated the mice with DFP at a dose of 75 mg/kg/d. Meanwhile, we treated the HT-22 cells with 100 μM DFP for half an hour and then infected cells with TgCtwh3in vitro. A qRT-PCR assay ofTgSAG1 levels showed a response to theT.gondiiburden. We used inductively coupled plasma mass spectrometry, an iron ion assay kit, Western blot analysis, glutathione and glutathione disulfide assay kits, a malonaldehyde assay kit, and immunofluorescence to detect the ferroptosis-related indexes in the mouse hippocampus and HT-22 cells. The inflammatory factors interferon-γ, tumor necrosis factor-α, transforming growth factor-β, and arginase 1 in the hippocampus and cells were detected using the Western blot assay. Hematoxylin and eosin staining, electron microscopy, and the Morris water maze experiment were used to evaluate the brain injuries of the mice. The results showed that TgCtwh3 infection is followed by the activation of ferroptosis-related signaling pathways and hippocampal pathological damage in mice. The use of DFP led to ferroptosis resistance and attenuated pathological changes, inflammatory reactions andT.gondiiburden of the mice, prolonging their survival time. The HT-22 cells with TgCtwh3 activated the ferroptosis pathway and was inhibit by DFPin vitro. In TgCtwh3-infected cells, inflammatory response and mitochondrial damage were severe, but these effects could be reduced by DFP. Our study elucidates the mechanism by whichT.gondiiinterferes with the host’s iron metabolism and activates ferroptosis, complementing the pathogenic mechanism of CT and further demonstrating the potential value of DFP for the treatment of CT.

Ocular toxoplasmosis (OT) is one of the most common causes of posterior uveitis. However, the pathogenic mechanisms of OT have not been well elucidated. Here, we used C57BL/6 (B6) mice to establish OT by peroral infection with 20 cysts of the TgCtWh6 strain, and severe ocular damage was observed by histopathological analysis in the eyes of infected mice. RNA-sequencing results showed that infection with T. gondii increased the expression of the NK-mediated cytotoxicity gene pathway at Day 30 after ocular T. gondii infection. Both NK-cell and CD49a⁺ NK-cell subsets are increased in ocular tissues, and the expression levels of LFA-1 in NK cells and ICAM-1 in the OT murine model were upregulated upon infection. Furthermore, inhibition of the interaction between LFA-1 and ICAM-1 with lifitegrast, a novel small molecule integrin antagonist, inhibited the protein expression of LFA-1 and ICAM-1 in murine OT and NK cells, improved the pathology of murine OT and influenced the secretion of cytokines in the OT murine model. In conclusion, the interaction between LFA-1 and ICAM-1 plays a role in the early regulation of the CD49a⁺ NK-cell proportion in an OT murine model. LFA-1/ ICAM-1 may be a key molecule in the pathogenesis of OT, and may provide new insights for potential immunotherapy.

Toxoplasma gondii (T. gondii), as an intracellular protozoan parasite, has the potential to disturb the homeostasis of trace metal elements in host cells. Zinc (Zn) is one of those essential metals that is required for combating infection. Zinc cellular homeostasis is controlled by zinc membrane transporters, including efflux and influx transporters. One of the Zrt-Irt-like protein (ZIP) transporters, ZIP8, facilitates zinc influx into the cytosol. It was recently reported to play significant roles in facilitating Zn uptakes during infection. Here, we investigated the function of ZIP8 in host defense against T. gondii infection in cultured alpha mouse liver 12 (AML12) hepatocytes and mice, with loss of ZIP8 function. Herein, C57BL/6 J female wild-type (WT) and ZIP8-KD mice (Slc39a8 knockdown mice), that were infected with tachyzoites of ToxoDB#9(TgCtwh3), were used as a model of acute toxoplasmosis. AML12 hepatocytes were transfected with lentivirus (LV), with silenced ZIP8 expression. Finally, we observed the function of hepatocytes pretreated with ZnCl₂ before TgCtwh3 infection in vivo and in vitro. In vivo, the levels of zinc ions and ZIP8 protein were upregulated after TgCtwh3 infection. ZIP8 knockdown exacerbated liver damage, further decreased antioxidant enzyme activity, promoted inflammatory mediator expression, and upregulated the rate of apoptosis. ZnCl₂ pretreatment before TgCtwh3 infection improved liver injury, increased antioxidant enzyme activity, restrained the expression of inflammatory mediators, and decreased the rate of apoptosis. The results in vitro were almost the same as those in vivo. This study defines the function of ZIP8-dependent zinc in hepatocyte damage during intracellular pathogen infection. Reagents that regulate ZIP8 activity might be developed as therapeutics to protect the liver function of toxoplasmosis.

Background: Toxoplasma gondii (T. gondii) reactivation is common, especially among immunocompromised individuals, such as AIDS patients. The cardiac involvement associated with toxoplasmosis, however, is usually obscured by neurological deterioration. The aim of this study was to observe the alterations in cardiac functions in various landmark periods after infection and to assess whether reactivation more seriously damages the heart. Methods: We established three infection models in mice using TgCtwh6, a major strain of T. gondii prevalent in China. The groups included an acute group, chronic latent group, and reactivation group. We evaluated the cardiac function impairment via H & E staining, Masson staining, echocardiography, myocardial enzyme profiles, and cardiac troponin, and detected the expression of inflammatory factors and antioxidant factors with Western blotting. Immunofluorescence was used to detect the expression of the macrophage marker F4/80. Results: Our results showed that damage to the heart occurred in the acute and reactivation groups. Impaired cardiac function manifested as a decrease in heart rate and a compensatory increase in left ventricular systolic function. Serum levels of cardiac enzymes also increased dramatically. In the chronic phase, myocardial fibrosis developed, diastolic functions became severely impaired, inflammation persisted, and macrophage expression was slightly reduced. Ultimately, reactivation infection exacerbated damage to cardiac function in mice, potentially leading to diastolic heart failure. Macrophages were strongly activated, and myocardial fibrosis was increased. In addition, the antioxidant capacity of the heart was severely affected by the infection. Conclusions: Taken together, these results suggested that the reactivation of T. gondii infection could aggravate injury to the heart, which could be associated with a host-cell-mediated immune response and strong cytokine production by macrophages, thus representing a novel insight into the pathogenic mechanism of toxoplasmosis.