Abstract

Abstract Primary human monocytes produce IL-12 and TNF in response to live Toxoplasma gondii tachyzoytes by a mechanism requiring parasite phagocytosis. Interestingly, in contrast to the patrolling CD16+ CD14+ subset, monocytes belonging to the major CD16neg CD14+ subset fail to secrete IL-12 and TNF following T. gondii exposure. Here we show that the CD16neg CD14+ subset, nevertheless, upregulates Il12b mRNA and thus is able to sense the parasite. In addition, we demonstrate that priming with IFNg, but not IFNa, enables CD16negCD14+ monocytes to produce IL-12 protein in response to T. gondii. This effect of IFNg priming was not due to augmented phagocytosis and was not associated with altered rates of glycolysis, oxygen consumption, or increased Il12b mRNA expression. To test the possible role of IFNγ on cytokine translation, we evaluated the requirement for mTOR (mTORC1/2) pathway, a key regulator of protein synthesis. The T. gondii-induced IL-12 response of IFNγ primed CD16neg monocytes was inhibited by Torin, an inhibitor of mTORC2, but surprisingly not by the classical mTORC1 inhibitor Rapamycin. Because mTORC2 has been shown to downregulate mTORC1 activity, we hypothesized that the priming effect of IFNg might result from the upregulation of mTORC2 resulting in suppressed mTORC1. Indeed, in the absence of IFNγ, suppression of mTORC1 in CD16neg monocytes with Rapamycin enhanced IL-12 secretion. Taken together, our results suggest that classical CD16neg CD14+ monocytes fail to secrete IL-12 in response to T. gondii due to increased mTORC1 activity, which can be reversed directly by Rapamycin or indirectly through stimulation of mTORC2 by IFNγ priming.

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