Barbara A. Butcher

Rapid Recruitment of Neutrophils Containing Prestored IL-12 During Microbial Infection

Neutrophils are well known to rapidly migrate to foci of infection, where they exert microbicidal functions. We sought to determine whether neutrophils responding to in vivo infection with the protozoan pathogen Toxoplasma gondii were capable of IL-12 production as suggested by recent in vitro studies. Intraperitoneal infection induced a neutrophil influx by 4 h, accompanied by ex vivo IL-12 p40 and p70 release. Approximately 85% of the neutrophils displayed intracellular stores of IL-12, as determined by flow cytometry and confocal fluorescence microscopy. Neutrophils from IFN-γ knockout mice also expressed IL-12, ruling out an IFN-γ-priming requirement. Neither infected nor uninfected peritoneal macrophages displayed intracellular IL-12, but these cells were strongly IL-10+. Infection per se was unnecessary for IL-12 production because peritoneal and peripheral blood neutrophils from uninfected animals contained IL-12+ populations. Expression of the granulocyte maturation marker Gr-1 (Ly-6G) was correlated with IL-12 production. Mice depleted of their granulocytes by mAb administration at the time of infection had decreased serum levels of IL-12 p40. These results suggest a model in which neutrophils with prestored IL-12 are rapidly mobilized to an infection site where they are triggered by the parasite to release cytokine. Our findings place neutrophils prominently in the cascade of early events leading to IL-12-dependent immunity to T. gondii.

Mouse neutrophils are professional antigen-presenting cells programmed to instruct Th1 and Th17 T-cell differentiation

Neutrophils play a major role in the innate immune system and are normally considered to be short-lived effector cells that exert anti-microbial activity and sometimes immunopathology. Here, we show that these cells possess an additional function as professional antigen-presenting cells capable of priming a T(h)1- and T(h)17-acquired immune response. Using flow cytometry, fluorescence microscopy and western blotting, we show that mouse neutrophils express MHC class II and co-stimulatory molecules CD80 and CD86 after T-cell co-incubation. Neutrophils pulsed with ovalbumin (OVA) process and present peptide antigen to OVA-specific T cells in an MHC class II-dependent manner. Importantly, we demonstrate that neutrophils can prime antigen-specific T(h)1 and T(h)17 immune responses even without the addition of exogenous cytokines to cell cultures.

Cutting Edge: IL-10-Independent STAT3 Activation by Toxoplasma gondii Mediates Suppression of IL-12 and TNF-α in Host Macrophages

Infection of mouse macrophages by Toxoplasma gondii renders the cells resistant to proinflammatory effects of LPS triggering. In this study, we show that cell invasion is accompanied by rapid and sustained activation of host STAT3. Activation of STAT3 did not occur with soluble T. gondii extracts or heat-killed tachyzoites, demonstrating a requirement for live parasites. Parasite-induced STAT3 phosphorylation and suppression of LPS-triggered TNF-α and IL-12 was intact in IL-10-deficient macrophages, ruling out a role for this anti-inflammatory cytokine in the suppressive effects of T. gondii. Most importantly, Toxoplasma could not effectively suppress LPS-triggered TNF-α and IL-12 synthesis in STAT3-deficient macrophages. These results demonstrate that T. gondii exploits host STAT3 to prevent LPS-triggered IL-12 and TNF-α production, revealing for the first time a molecular mechanism underlying the parasite’s suppressive effect on macrophage proinflammatory cytokine production.

Toxoplasma gondii Rhoptry Kinase ROP16 Activates STAT3 and STAT6 Resulting in Cytokine Inhibition and Arginase-1-Dependent Growth Control

The ROP16 kinase of Toxoplasma gondii is injected into the host cell cytosol where it activates signal transducer and activator of transcription (STAT)-3 and STAT6. Here, we generated a ROP16 deletion mutant on a Type I parasite strain background, as well as a control complementation mutant with restored ROP16 expression. We investigated the biological role of the ROP16 molecule during T. gondii infection. Infection of mouse bone marrow-derived macrophages with rop16-deleted (ΔROP16) parasites resulted in increased amounts of IL-12p40 production relative to the ROP16-positive RH parental strain. High level IL-12p40 production in ΔROP16 infection was dependent on the host cell adaptor molecule MyD88, but surprisingly was independent of any previously recognized T. gondii triggered pathway linking to MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1ß and IL-18). In addition, ROP16 was found to mediate the suppressive effects of Toxoplasma on LPS-induced cytokine synthesis in macrophages and on IFN-γ-induced nitric oxide production by astrocytes and microglial cells. Furthermore, ROP16 triggered synthesis of host cell arginase-1 in a STAT6-dependent manner. In fibroblasts and macrophages, failure to induce arginase-1 by ΔROP16 tachyzoites resulted in resistance to starvation conditions of limiting arginine, an essential amino acid for replication and virulence of this parasite. ΔROP16 tachyzoites that failed to induce host cell arginase-1 displayed increased replication and dissemination during in vivo infection. We conclude that encounter between Toxoplasma ROP16 and the host cell STAT signaling cascade has pleiotropic downstream effects that act in multiple and complex ways to direct the course of infection.

p47 GTPases regulate Toxoplasma gondii survival in activated macrophages.

ABSTRACT The cytokine gamma interferon (IFN-γ) is critical for resistance to Toxoplasma gondii. IFN-γ strongly activates macrophages and nonphagocytic host cells to limit intracellular growth of T. gondii; however, the cellular factors that are required for this effect are largely unknown. We have shown previously that IGTP and LRG-47, members of the IFN-γ-regulated family of p47 GTPases, are required for resistance to acute T. gondii infections in vivo. In contrast, IRG-47, another member of this family, is not required. In the present work, we addressed whether these GTPases are required for IFN-γ-induced suppression of T. gondii growth in macrophages in vitro. Bone marrow macrophages that lacked IGTP or LRG-47 displayed greatly attenuated IFN-γ-induced inhibition of T. gondii growth, while macrophages that lacked IRG-47 displayed normal inhibition. Thus, the ability of the p47 GTPases to limit acute infection in vivo correlated with their ability to suppress intracellular growth in macrophages in vitro. Using confocal microscopy and sucrose density fractionation, we demonstrated that IGTP largely colocalizes with endoplasmic reticulum markers, while LRG-47 was mainly restricted to the Golgi. Although both IGTP and LRG-47 localized to vacuoles containing latex beads, neither protein localized to vacuoles containing live T. gondii. These results suggest that IGTP and LRG-47 are able to regulate host resistance to acute T. gondii infections through their ability to inhibit parasite growth within the macrophage.

p38 MAPK autophosphorylation drives macrophage IL-12 production during intracellular infection.

The intracellular protozoan Toxoplasma gondii triggers rapid MAPK activation in mouse macrophages (Mφ). We used synthetic inhibitors and dominant-negative Mφ mutants to demonstrate that T. gondii triggers IL-12 production in dependence upon p38 MAPK. Chemical inhibition of stress-activated protein kinase/JNK showed that this MAPK was also required for parasite-triggered IL-12 production. Examination of upstream MAPK kinases (MKK) 3, 4, and 6 that function as p38 MAPK activating kinases revealed that parasite infection activates only MKK3. Nevertheless, in MKK3−/− Mφ, p38 MAPK activation was near normal and IL-12 production was unaffected. Recently, MKK-independent p38α MAPK activation via autophosphorylation was described. Autophosphorylation depends upon p38α MAPK association with adaptor protein, TGF-β-activated protein kinase 1-binding protein-1. We observed TGF-β-activated protein kinase 1-binding protein-1-p38α MAPK association that closely paralleled p38 MAPK phosphorylation during Toxoplasma infection of Mφ. Furthermore, a synthetic p38 catalytic-site inhibitor blocked tachyzoite-induced p38α MAPK phosphorylation. These data are the first to demonstrate p38 MAPK autophosphorylation triggered by intracellular infection.

Toxoplasma gondii Triggers Myeloid Differentiation Factor 88-Dependent IL-12 and Chemokine Ligand 2 (Monocyte Chemoattractant Protein 1) Responses Using Distinct Parasite Molecules and Host Receptors

Toll-like receptors (TLR) that signal through the common adaptor molecule myeloid differentiation factor 88 (MyD88) are essential in proinflammatory cytokine responses to many microbial pathogens. In this study we report that Toxoplasma gondii triggers neutrophil IL-12 and chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1) production in strict dependence upon functional MyD88. Nevertheless, the responses are distinct. Although we identify TLR2 as the receptor triggering CCL2 production, parasite-induced IL-12 release did not involve this TLR. The production of both IL-12 and CCL2 was increased after neutrophil activation with IFN-γ. However, the synergistic effect of IFN-γ on IL-12, but not CCL2, was dependent upon Stat1 signal transduction. Although IL-10 was a potent down-regulator of Toxoplasma-triggered neutrophil IL-12 release, the cytokine had no effect on parasite-induced CCL2 production. Soluble tachyzoite Ag fractionation demonstrated that CCL2- and IL-12 inducing activities are biochemically distinct. Importantly, Toxoplasma cyclophilin-18, a molecule previously shown to induce dendritic cell IL-12, was not involved in neutrophil IL-12 production. Our results show for the first time that T. gondii possesses multiple molecules triggering distinct MyD88-dependent signaling cascades, that these pathways are independently regulated, and that they lead to distinct profiles of cytokine production.

STAT1 is essential for antimicrobial effector function but dispensable for gamma interferon production during Toxoplasma gondii infection.

ABSTRACT The opportunistic protozoan Toxoplasma gondii is a prototypic Th1-inducing pathogen inducing strong gamma interferon (IFN-γ) cytokine responses that are required to survive infection. Intracellular signaling intermediate STAT1 mediates many effects of IFN-γ and is implicated in activation of T-bet, a master regulator of Th1 differentiation. Here, we show that T. gondii-infected STAT1-null mice fail to upregulate the IFN-γ-dependent effector molecules inducible nitric oxide synthase (iNOS), IGTP, and LRG-47, which are required for mice to survive infection. Both T-bet and interleukin-12 receptor β2 (IL-12Rβ2) failed to undergo normal upregulation in response to T. gondii. Development of IFN-γ-producing CD4+ and CD8+ T lymphocytes was severely curtailed in the absence of STAT1, but a substantial level of STAT1-independent non-T-cell-derived IFN-γ was induced. Absence of STAT1 also resulted in increased IL-4, Arg1, Ym1, and Fizz1, markers of Th2 differentiation and alternative macrophage activation. Together, the results show that T. gondii induces STAT1-dependent T-lymphocyte and STAT1-independent non-T-cell IFN-γ production, but that effector functions of this type 1 cytokine cannot operate in the absence of STAT1, resulting in extreme susceptibility to acute infection.

Mechanism of Entry Determines the Ability of Toxoplasma gondii To Inhibit Macrophage Proinflammatory Cytokine Production

ABSTRACT Macrophages (Mφ) infected with tachyzoites of the opportunistic protozoan Toxoplasma gondii are blocked in production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12) in response to lipopolysaccharide (LPS) triggering, and this is associated with parasite-induced inhibition of NFκB translocation. Here, we demonstrate a requirement for active invasion in the ability of the parasite to mediate suppression. Neither soluble tachyzoite antigen nor secreted products were suppressive, and heat-inactivated, antibody-coated tachyzoites, which efficiently entered the cell through receptor-mediated uptake, failed to inhibit LPS responses. Cytochalasin D, a drug blocking tachyzoite invasion of, but not adherence to, Mφ, severely curtailed Toxoplasma-induced suppression. In addition, parasite-induced nonresponsiveness, as measured by TNF-α production, was reversed by treating infected cells with the toxoplasmastatic drugs pyrimethamine and 6-thioxanthine prior to LPS stimulation. A divergence in IL-12 and TNF-α responses was found during extended incubation of tachyzoites and Mφ in that 24 h of incubation of infected Mφ resulted in IL-12, but not TNF-α, secretion, and production of the latter cytokine remained suppressed when these cells were subjected to LPS triggering. Our results demonstrate that active invasion and survival of the parasite within the parasitophorous vacuole are required to induce and maintain Mφ cytokine-specific nonresponsiveness to LPS. They also show that the effects of Toxoplasma on IL-12 and TNF-α production are nonidentical, with the parasite exerting a longer-lasting suppression of the latter.

Toxoplasma gondii Interferes with Lipopolysaccharide-Induced Mitogen-Activated Protein Kinase Activation by Mechanisms Distinct from Endotoxin Tolerance

We show in this study that Toxoplasma gondii infection induces rapid activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2, and stress-activated protein kinase/c-Jun N-terminal kinase MAPK, followed promptly by their deactivation in mouse macrophages. Nevertheless, when infected cells were subsequently subjected to LPS triggering, MAPK activation was severely defective, in particular in the case of p38 MAPK, which is required for LPS-triggered TNF-α and IL-12 production. Similar effects occurred during endotoxin tolerance, but the phenomena were distinct. LPS pretriggering failed to activate the major p38 MAPK kinase, MAPK kinase 3/6. Toxoplasma infection, in contrast, resulted in sustained activation of this kinase. Furthermore, endotoxin pre-exposure blocked IκBα degradation upon subsequent LPS triggering, but this was not the case for Toxoplasma preinfection. Endotoxin-mediated down-regulation of the LPS receptor, Toll-like receptor 4, has been suggested as one possible mechanism contributing to tolerance, and we found in this study that LPS down-modulated Toll-like receptor 4 expression. In contrast, Toxoplasma infection induced up-regulation of this pattern recognition receptor. Our results show that T. gondii blocks LPS-triggered cytokine production in part through MAPK inactivation, and that this occurs through pathways distinct from endotoxin-induced tolerance.