UC Irvine

Toxoplasma gondii is an obligate intracellular eukaryotic parasite that is estimated to infect one-third of the global population and is especially life-threatening in developing fetuses and immunocompromised individuals. Innate immune cells contribute to host defense against T. gondii infection. Specifically, monocytes are rapidly recruited to sites of infection and CCR2 or CCL2 KO mice are more susceptible to T. gondii infection. Monocytes protect against infection by initiating a robust inflammatory response which is in part mediated by release of IL-1β during activation of the NLRP3 inflammasome. IL-1β is also implicated in the development of many autoimmune disorders like rheumatoid arthritis and atherosclerosis, but there is still much that is unknown about how human monocytes produce, process and release IL-1β. We have identified that T. gondii induces IL-1β production via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1β release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced transcription and production of pro-IL-1β protein. Inhibition of PKCδ, CARD9/MALT-1 and IKK also reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knock-out (KO) of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1β protein levels and IL-1β release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1β transcriptional activation. We have also found that primary human monocytes treated with a caspase-8 inhibitor released less IL-1β after T. gondii infection than control cells. Similarly, caspase-1 and caspase-8 KO human monocytic THP-1 cells, but not caspase-4 or -5 KO cells were impaired in their release of IL-1β after infection compared to empty vector (EV) THP-1 cells. We found caspase-8 deficiency did not significantly affect the pro-IL-1β transcripts or production of pro-IL-1β protein. Similarly, caspase-8 had no significant affect on NLRP3 inflammasome activation or cleavage of pro-IL-1β to mature IL-1β during T. gondii infection. Instead, caspase-8 deficiency appeared to stunt the release mechanism of IL-1β from infected cells as mature-IL-1β would accumulate intracellularly in these KO cells. IL-1β release from T. gondii-infected primary human monocytes did require an NLRP3-ASC-caspase-1 inflammasome. While the release mechanism of IL-1β during NLRP3 inflammasome activation often requires cleavage of gasdermin D (GSDMD), formation of pores in the cell membrane and induction of an inflammatory form of cell death known as pyroptosis, human monocytes released IL-1β independent of these factors. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1β in a cell death- and GSDMD-independent manner. This research also describes a novel role for caspase-8 in IL-1β release from T. gondii-infected monocytes and contributes to the growing notion that IL-1β can be released from human myeloid cells independent of cell death. Collectively, this research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection.