Dual transcriptomic analysis of Toxoplasma gondii infection in a human PBMC ex vivo model

The mechanisms governing host-parasite interactions in human toxoplasmosis remain insufficiently characterized, as research has relied on murine models that fail to capture human cellular responses. Murine resistance to Toxoplasma gondii depends on the IL-12/IFN-γ axis and immunity-related GTPases (IRGs); however, these differ in humans due to the absence of TLR11/12 and a distinct IRG repertoire. We implemented the EXMOWS (ex vivo model without supplements) to investigate early human host-parasite interactions without the biological artifacts induced by fetal bovine serum (FBS) or cryopreservation. Peripheral blood mononuclear cells (PBMCs) were freshly isolated from five healthy individuals (three Toxoplasma IgG+, two seronegative) and infected with T. gondii (RH strain; multiplicity of infection, 1:3) in supplement-free media. Global transcriptional profiling was performed using dual RNA-seq at 0, 1, and 6 h post-infection (hpi). We identified differentially expressed host genes (DEGs), characterized by potent early activation of innate immune sensing, nuclear factor-κB (NF-κB) signalling, and type I/II interferon signalling pathways. Key overexpressed hubs included IL1B, IL1A, CXCL8, IL6, and TNF, whereas NFBIA and IL10 were significantly downregulated. Simultaneously, T. gondii modulated hundreds of genes, including major virulence factors, such as ROP16, ROP18, GRA7, and GRA15. The EXMOWS model reveals that human primary cells initiate a robust transcriptional Th1 and NF-κB response within 6 h of infection, potentially preceding or overcoming early parasite-mediated suppressive mechanisms. These results provide a standardized, high-resolution framework for identifying protective molecular signatures in human toxoplasmosis.