Research Article: Toxoplasma gondii activates a Syk-CARD9-NF-κB signaling axis and gasdermin D-independent release of IL-1β during infection of primary human monocytes

Date Published: August 26, 2019

Publisher: Public Library of Science

Author(s): William J. Pandori, Tatiane S. Lima, Sharmila Mallya, Tiffany H. Kao, Lanny Gov, Melissa B. Lodoen, Eric Y Denkers.

http://doi.org/10.1371/journal.ppat.1007923

Abstract

IL-1β is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1β; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1β 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 IL-1β transcripts and the production of pro-IL-1β. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knockout 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. IL-1β release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1β to wild-type THP-1 cells after T. gondii infection. 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 expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection.

Partial Text

Toxoplasma gondii is an obligate intracellular foodborne parasite capable of infecting and replicating in any nucleated cell of its infected hosts [1]. Global estimates suggest that as much as a third of the world population is chronically infected with this parasite and that over thirty million people become ill from T. gondii infections each year [2,3]. While a robust immune response typically controls the infection, T. gondii poses severe health risks to immunocompromised individuals and to the developing fetus during congenital disease [4,5]. In particular, CD4+ and CD8+ T cells and the production of IFN-γ are required for protection against T. gondii infection [6,7]. Innate immune cells also contribute significantly to host defense against T. gondii through the production of IL-12 and cell intrinsic mechanisms of host defense [8]. Monocytes, in particular, are among the first cells recruited to sites of T. gondii infection and are critical for parasite control during both the acute and chronic stages of disease [9–14].

Syk is a tyrosine kinase expressed in immune cells and is typically activated by receptors or receptor-associated adaptor proteins with cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs) [49]. Syk is known to be critical for lymphatic development, inflammatory signaling, and inflammasome activation [50]. We now demonstrate that inhibition of Syk in primary human monocytes and genetic deletion of Syk in monocytic THP-1 cells both significantly reduced IL-1β transcripts and pro-IL-1β production during T. gondii infection, indicating a role for Syk in IL-1β synthesis in parasite-infected cells. Among other signaling pathways, Syk can signal through PKCδ and CARD9 to induce NF-κB activation [46,51], and indeed, inhibitors against PKCδ, CARD9/MALT-1, and IKK, or genetic deletion of PKCδ and CARD9 in THP-1 cells revealed the importance of this pathway in IL-1β synthesis in T. gondii-infected monocytes, as depicted in Fig 8. Syk also contributed to NLRP3 transcript induction in response to T. gondii infection, further supporting a role for Syk in the priming of both IL-1β and NLRP3. In contrast, Syk appeared to be less important for the production of pro-IL-1β in LPS-stimulated primary human monocytes, but rather, seemed to contribute more substantially to IL-1β release from these cells. LPS-stimulated monocytes likely rely more heavily on canonical NF-κB signaling downstream of TLR4 and MyD88 for IL-1β transcription, and on Syk for processing and release of IL-1β. Indeed, it has been shown that Syk can lead to activation of the NLRP3 inflammasome through the indirect phosphorylation of the inflammasome adaptor protein ASC [45,52]. ASC phosphorylation induces its oligomerization, facilitating activation of the NLRP3 inflammasome and caspase-1 [53,54]. Collectively, our data indicate that immune cells responding to pathogens harboring multiple PAMPs or vitaPAMPs will likely regulate IL-1β differently than cells responding to a single PAMP or stimulus. For example, primary human monocytes utilize Syk signaling through ERK1/2 to produce IL-1β when dengue virus is in complex with antibody [43], and our research shows that during T. gondii infection Syk activates a separate signaling pathway to reach the same net result. Thus, examining the production and processing of IL-1β and other cytokines during infection with various live pathogens may unveil new pathways of regulation that could be critical for enhancing or dampening inflammation during different types of infections.

 

Source:

http://doi.org/10.1371/journal.ppat.1007923

 

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