Search

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.

Activation of the NADPH oxidase enzyme complex results in the production of the oxygen free radical, superoxide anion (O2.-). Superoxide anion is critical for host defense against fungal and bacterial pathogens and efficient immune responses; however, uncontrolled monocyte-derived O2.- may contribute to chronic inflammation and tissue injury. We have previously identified several pathways that regulate the activity of NADPH oxidase in human monocytes; however, the receptor(s) responsible for the activation of NADPH oxidase in primary human monocytes have not yet been determined. This study shows that pattern recognition receptors, namely Dectin-1 and Complement Receptor 3, are essential for regulating NADPH oxidase activity in Zymosan-activated human monocytes. We show that TLR2 and TLR4 are not required for NADPH oxidase activation by Zymosan. In addition this study focuses on Dectin-1 downstream signaling and complex formation with intracellular signaling proteins. Our findings in human monocytes are supportive of the prior recognized role of Src and Syk tyrosine kinases in regulating Dectin-1-mediated ROS production in murine macrophages. Our data also shows that Src and Syk are tyrosine phosphorylated in Zymosan- treated cells and that they both regulate each others activity. Furthermore, we focused on Dectin-1 complex formation with intracellular signaling proteins including Syk, Src and PKCδ, protein kinases that regulates NADPH oxidase activity in human monocytes. This is the first study to show the involvement of PKCδ in Dectin-1 signaling. We found that the activity of PKCδ is required for its own complex formation with Dectin-1 as well as Syk-Dectin-1 interaction. In contrast, Src and Syk inhibitors had no effect on PKCδ association with Dectin-1. Blocking the activity of Src inhibited phospho-Syk/Dectin-1 complex formation which supports the role of Src in regulating Syk tyrosine phosphorylation/activation. Our data confirms that Dectin-1, a pattern recognition receptor, is a key player in the regulation of NADPH oxidase in Zymosan-activated human monocytes and we introduce PKCδ as a novel player in Dectin-1 signaling.To expand on this project, the study also includes data reporting an endogenous pathophysiological protein, osteopontin, as a novel ligand for Dectin-1. We show here that osteopontin induces the oxidative burst in monocytes through Dectin-1. We also show data supporting binding of osteopontin to Dectin-1. Taken together, our study provides new insights into Dectin-1 ligands and downstream signaling in primary human monocytes and highlights novel signaling pathways utilized in these important cells. Our findings are relevant for understanding the regulation of NADPH oxidase in the innate immune response and in chronic inflammatory diseases.

Differential-display or RNA fingerprint was applied to identify genes differentially expressed in monocyte maturation induced by an immunomodulating peptide on human peripheral blood mononuclear cells. Two unknown sequences (06c22 and 06c71) and p21 protein (cyclin dependent kinase inhibitor) were repressed, and three genes activated: Cathepsin D, DRP2 (dihydropirimidinase related protein 2), and gp91phox (91-kDa subunit of citochrome b(558)). Phenotype of evolving monocytes was analyzed by flow cytometry and mRNA level of identified genes determined by reverse transcription-PCR. The expression pattern of identified genes seemed to correlate with different monocyte subsets, monocyte-derived cells, and expected functional changes. After peptide addition, immature monocytes were initially activated, increasing the expression of CD25, CD69, and HLA-DR markers. This was accompanied by repression of p21 and the two unknown sequences, along with the simultaneous activation of Cathepsin D and DRP2. Later, the differentiation marker CD16 rose, and gp91phox gene expression activated. Further maturation led certain monocytes to express marker CD23 and gp91phox expression to reach a maximum, while Cathepsin D and DRP2 dropped to preactivation levels. Results reflect part of the evolution of immature monocytes toward macrophages and monocyte-derived dendritic cell precursors.

Activated and differentiated human monocytes with a CD14+CD16+ phenotype were found to contain a DNase activity associated with secretion granules. Activated cells were obtained from patients with autoimmune diseases. Activation and differentiation of monocytes were also achieved after incubation of PBMC from healthy subjects with protein A (SpA) or immunopotentiating peptides. DNase activity corresponded to a 66-kDa protein, similar to that described in granules from T lymphocytes, active preferentially on double-strand DNA. DNA fragmentation activity increased when NO donors were present; the activity was higher in the presence of Ca2+, and at low pH values. The Ca2+-dependent activity was inhibited by Zn2+. NO-dependent activity was additive with that of Ca2+-dependent and it was not inhibited by Zn2+. Dithiothreitol did not modify the effect of NO on DNase activity. Incubation of PBMC in the presence of NMLA, an inhibitor of NO synthases, decreased this DNase activity. Data reported clearly suggest a regulatory role of NO in granule-associated DNase activity