Unraveling host and parasite pathways in human innate immunity to Toxoplasma gondii

Toxoplasma gondii is a pathogen of global importance. Innate immunity is critical for control of acute infection. In vivo mouse models have shown that monocytes are rapidly recruited to sites of T. gondii infection and MCP-1 and CCR2 knock-out mice that fail to do so succumb to infection, underscoring the importance of monocytes in host defense. However, the role of human monocytes in immunity to T. gondii and their specific interactions with T. gondii are not well understood.Human monocytes are actively infected by T. gondii and permissive to parasite replication. Infected human monocytes release interleukin-1beta (IL-1beta), a "master regulator" of inflammation that has been shown to be protective against T. gondii in vivo. However, the host and parasite factors regulating T. gondii-induced IL-1beta production in human monocytes were unknown. We found that T. gondii induces IL-1beta transcript, post-translational processing, and release from human monocytes. We demonstrate a role for host inflammasome components caspase-1 and ASC and the parasite protein GRA15 in the induction and release of IL-1beta. This work provides important mechanistic insight into the regulation of a key mediator of inflammation and is the first to identify a T. gondii factor that drives innate immune responses in human cells.Recently, there has been increasing appreciation for the heterogeneity of circulating monocytes. Three phenotypically and functionally distinct subpopulations have been defined in humans based on CD14 and CD16 expression. However, the interactions of specific monocyte subsets with T. gondii and their functional outcomes have not been elucidated. We found that T. gondii preferentially invades classical (CD14+CD16-) and intermediate (CD14+CD16+) monocytes and that these subsets are more permissive to intracellular parasite survival. All monocyte subsets were capable of phagocytosing and degrading Mycalolide B-treated parasites. T. gondii infection induces IL-1beta in all three monocyte subsets, but the resident monocytes (CD14loCD16+) showed an enhanced IL-1beta response. These data suggest that resident monocytes are less permissive to infection and may play a critical role in parasite control. Collectively, our work defines host and parasite pathways driving innate immunity and contributes to a better understanding of the role of human monocytes in parasite control

Evasion of Human Neutrophil-Mediated Host Defense during Toxoplasma gondii Infection

Neutrophils are a major player in host immunity to infection; however, the mechanisms by which human neutrophils respond to the intracellular protozoan parasite Toxoplasma gondii are still poorly understood. In the current study, we found that, whereas primary human monocytes produced interleukin-1beta (IL-1β) in response to T. gondii infection, human neutrophils from the same blood donors did not. Moreover, T. gondii inhibited lipopolysaccharide (LPS)-induced IL-1β synthesis in human peripheral blood neutrophils. IL-1β suppression required active parasite invasion, since heat-killed or mycalolide B-treated parasites did not inhibit IL-1β release. By investigating the mechanisms involved in this process, we found that T. gondii infection of neutrophils treated with LPS resulted in reduced transcript levels of IL-1β and NLRP3 and reduced protein levels of pro-IL-1β, mature IL-1β, and the inflammasome sensor NLRP3. In T. gondii-infected neutrophils stimulated with LPS, the levels of MyD88, TRAF6, IKKα, IKKβ, and phosphorylated IKKα/β were not affected. However, LPS-induced IκBα degradation and p65 phosphorylation were reduced in T. gondii-infected neutrophils, and degradation of IκBα was reversed by treatment with the proteasome inhibitor MG-132. Finally, we observed that T. gondii inhibited the cleavage and activity of caspase-1 in human neutrophils. These results indicate that T. gondii suppression of IL-1β involves a two-pronged strategy whereby T. gondii inhibits both NF-κB signaling and activation of the NLRP3 inflammasome. These findings represent a novel mechanism of T. gondii evasion of human neutrophil-mediated host defense by targeting the production of IL-1β.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that infects approximately one-third of humans worldwide and can invade virtually any nucleated cell in the human body. Although it is well documented that neutrophils infiltrate the site of acute T. gondii infection, there is limited understanding of how human neutrophils respond to T. gondii Neutrophils control infectious pathogens by a variety of mechanisms, including the release of the cytokine IL-1β, a major driver of inflammation during infection. This study reveals that T. gondii is able to inhibit IL-1β production in human neutrophils by impairing the activation of the NF-κB signaling pathway and by inhibiting the inflammasome, the protein complex responsible for IL-1β maturation. This two-pronged strategy of targeting the IL-1β pathway may facilitate the survival and spread of T. gondii during acute infection

Evasion of Human Neutrophil-Mediated Host Defense during Toxoplasma gondii Infection.

Neutrophils are a major player in host immunity to infection; however, the mechanisms by which human neutrophils respond to the intracellular protozoan parasite Toxoplasma gondii are still poorly understood. In the current study, we found that, whereas primary human monocytes produced interleukin-1beta (IL-1β) in response to T. gondii infection, human neutrophils from the same blood donors did not. Moreover, T. gondii inhibited lipopolysaccharide (LPS)-induced IL-1β synthesis in human peripheral blood neutrophils. IL-1β suppression required active parasite invasion, since heat-killed or mycalolide B-treated parasites did not inhibit IL-1β release. By investigating the mechanisms involved in this process, we found that T. gondii infection of neutrophils treated with LPS resulted in reduced transcript levels of IL-1β and NLRP3 and reduced protein levels of pro-IL-1β, mature IL-1β, and the inflammasome sensor NLRP3. In T. gondii-infected neutrophils stimulated with LPS, the levels of MyD88, TRAF6, IKKα, IKKβ, and phosphorylated IKKα/β were not affected. However, LPS-induced IκBα degradation and p65 phosphorylation were reduced in T. gondii-infected neutrophils, and degradation of IκBα was reversed by treatment with the proteasome inhibitor MG-132. Finally, we observed that T. gondii inhibited the cleavage and activity of caspase-1 in human neutrophils. These results indicate that T. gondii suppression of IL-1β involves a two-pronged strategy whereby T. gondii inhibits both NF-κB signaling and activation of the NLRP3 inflammasome. These findings represent a novel mechanism of T. gondii evasion of human neutrophil-mediated host defense by targeting the production of IL-1β.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that infects approximately one-third of humans worldwide and can invade virtually any nucleated cell in the human body. Although it is well documented that neutrophils infiltrate the site of acute T. gondii infection, there is limited understanding of how human neutrophils respond to T. gondii Neutrophils control infectious pathogens by a variety of mechanisms, including the release of the cytokine IL-1β, a major driver of inflammation during infection. This study reveals that T. gondii is able to inhibit IL-1β production in human neutrophils by impairing the activation of the NF-κB signaling pathway and by inhibiting the inflammasome, the protein complex responsible for IL-1β maturation. This two-pronged strategy of targeting the IL-1β pathway may facilitate the survival and spread of T. gondii during acute infection

Evasion of Human Neutrophil-Mediated Host Defense during Toxoplasma gondii Infection.

Neutrophils are a major player in host immunity to infection; however, the mechanisms by which human neutrophils respond to the intracellular protozoan parasite Toxoplasma gondii are still poorly understood. In the current study, we found that, whereas primary human monocytes produced interleukin-1beta (IL-1β) in response to T. gondii infection, human neutrophils from the same blood donors did not. Moreover, T. gondii inhibited lipopolysaccharide (LPS)-induced IL-1β synthesis in human peripheral blood neutrophils. IL-1β suppression required active parasite invasion, since heat-killed or mycalolide B-treated parasites did not inhibit IL-1β release. By investigating the mechanisms involved in this process, we found that T. gondii infection of neutrophils treated with LPS resulted in reduced transcript levels of IL-1β and NLRP3 and reduced protein levels of pro-IL-1β, mature IL-1β, and the inflammasome sensor NLRP3. In T. gondii-infected neutrophils stimulated with LPS, the levels of MyD88, TRAF6, IKKα, IKKβ, and phosphorylated IKKα/β were not affected. However, LPS-induced IκBα degradation and p65 phosphorylation were reduced in T. gondii-infected neutrophils, and degradation of IκBα was reversed by treatment with the proteasome inhibitor MG-132. Finally, we observed that T. gondii inhibited the cleavage and activity of caspase-1 in human neutrophils. These results indicate that T. gondii suppression of IL-1β involves a two-pronged strategy whereby T. gondii inhibits both NF-κB signaling and activation of the NLRP3 inflammasome. These findings represent a novel mechanism of T. gondii evasion of human neutrophil-mediated host defense by targeting the production of IL-1β.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that infects approximately one-third of humans worldwide and can invade virtually any nucleated cell in the human body. Although it is well documented that neutrophils infiltrate the site of acute T. gondii infection, there is limited understanding of how human neutrophils respond to T. gondii Neutrophils control infectious pathogens by a variety of mechanisms, including the release of the cytokine IL-1β, a major driver of inflammation during infection. This study reveals that T. gondii is able to inhibit IL-1β production in human neutrophils by impairing the activation of the NF-κB signaling pathway and by inhibiting the inflammasome, the protein complex responsible for IL-1β maturation. This two-pronged strategy of targeting the IL-1β pathway may facilitate the survival and spread of T. gondii during acute infection

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

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

Epstein-Barr virus-induced gene 3 negatively regulates neuroinflammation and T cell activation following coronavirus-induced encephalomyelitis.

Epstein-Barr virus-induced gene 3 (EBI3) associates with p28 and p35 to form the immunomodulatory cytokines IL-27 and IL-35, respectively. Infection of EBI3-/- mice with the neuroadapted JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality that was not associated with impaired ability to control viral replication but enhanced T cell and macrophage infiltration into the CNS. IFN-γ secretion from virus-specific CD4+ and CD8+ T cells isolated from infected EBI3-/- mice was augmented while IL-10 expression muted in comparison to infected WT mice. These data demonstrate a regulatory role for EBI3-associated cytokines in controlling host responses following CNS viral infection

Type II Toxoplasma gondii Induction of CD40 on Infected Macrophages Enhances Interleukin-12 Responses

Toxoplasma gondii is an obligate intracellular parasite that can cause severe neurological disease in infected humans. CD40 is a receptor on macrophages that plays a critical role in controlling T. gondii infection. We examined the regulation of CD40 on the surface of T. gondii-infected bone marrow-derived macrophages (BMdMs). T. gondii induced CD40 expression both at the transcript level and on the cell surface, and interestingly, the effect was parasite strain specific: CD40 levels were dramatically increased in type II T. gondii-infected BMdMs compared to type I- or type III-infected cells. Type II induction of CD40 was specific to cells harboring intracellular parasites and detectable as early as 6 h postinfection (hpi) at the transcript level. CD40 protein expression peaked at 18 hpi. Using forward genetics with progeny from a type II × type III cross, we found that CD40 induction mapped to a region of chromosome X that included the gene encoding the dense granule protein 15 (GRA15). Using type I parasites stably expressing the type II allele of GRA15 (GRA15(II)), we found that type I GRA15(II) parasites induced the expression of CD40 on infected cells in an NF-κB-dependent manner. In addition, stable expression of hemagglutinin-tagged GRA15(II) in THP-1 cells resulted in CD40 upregulation in the absence of infection. Since CD40 signaling contributes to interleukin-12 (IL-12) production, we examined IL-12 from infected macrophages and found that CD40L engagement of CD40 amplified the IL-12 response in type II-infected cells. These data indicate that GRA15(II) induction of CD40 promotes parasite immunity through the production of IL-12

MicroRNA profiling identifies miR-34a and miR-21 and their target genes JAG1 and WNT1 in the coordinate regulation of dendritic cell differentiation

MicroRNAs (miRNAs, miRs) modulate a multitude of cellular events. Here, we identify functional miRNA-protein networks that regulate human monocyte-derived dendritic cell (MDDC) differentiation. miRNA profiling revealed stage-specific differential expression of 20 miRNAs during days 1, 3, and 5 of MDDC differentiation. To identify and prioritize miRNA-protein networks for functional validation, we developed a target ranking algorithm that incorporates many features of miRNA regulatory networks. This system prioritized miR-21, miR-34a, and their cognate targets WNT1 and JAG1 for functional validation. Inhibition of both miR-21 and miR-34a stalled MDDC differentiation, as quantified by DC-SIGN/CD14 expression ratios, showing cooperative involvement of these miRNAs in MDDC differentiation. We confirmed that the 3′ untranslated regions of WNT1 and JAG1 were functional targets of these miRNAs and provide evidence that these targets were translationally suppressed. Significantly, exogenously added Wnt-1 and Jagged-1 also stalled MDDC differentiation, suggesting that miRNA-mediated inhibition of endogenous WNT1 and JAG1 expression was important for proper MDDC differentiation. Finally, inhibition of miR-21 and miR-34a, or addition of Wnt-1 and Jagged-1, led to a decrease in endocytic capacity, a key function of immature DCs. Thus, our novel approach identified and validated some miRNA-protein networks involved in phenotypic and functional MDDC differentiation