Mechanism of entry determines the ability of Toxoplasma gondii to inhibit macrophage proinflammatory cytokine production

Macrophages (Mphi) infected with tachyzoites of the opportunistic protozoan Toxoplasma gondii are blocked in production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-12 (IL-12) in response to lipopolysaccharide (LPS) triggering, and this is associated with parasite-induced inhibition of NFkappaB translocation. Here, we demonstrate a requirement for active invasion in the ability of the parasite to mediate suppression. Neither soluble tachyzoite antigen nor secreted products were suppressive, and heat-inactivated, antibody-coated tachyzoites, which efficiently entered the cell through receptor-mediated uptake, failed to inhibit LPS responses. Cytochalasin D, a drug blocking tachyzoite invasion of, but not adherence to, Mphi, severely curtailed Toxoplasma-induced suppression. In addition, parasite-induced nonresponsiveness, as measured by TNF-alpha production, was reversed by treating infected cells with the toxoplasmastatic drugs pyrimethamine and 6-thioxanthine prior to LPS stimulation. A divergence in IL-12 and TNF-alpha responses was found during extended incubation of tachyzoites and Mphi in that 24 h of incubation of infected Mphi resulted in IL-12, but not TNF-alpha, secretion, and production of the latter cytokine remained suppressed when these cells were subjected to LPS triggering. Our results demonstrate that active invasion and survival of the parasite within the parasitophorous vacuole are required to induce and maintain Mphi cytokine-specific nonresponsiveness to LPS. They also show that the effects of Toxoplasma on IL-12 and TNF-alpha production are nonidentical, with the parasite exerting a longer-lasting suppression of the latter.