Our previous studies showed that TLR11 recognizes a profilin protein from T. gondii (TgPRF) and is required for optimal production of p40 IL-12as well as optimal control of infection. We have now extended these observations and in collaboration with Dr. Sankar Gosh (Columbia University, NY) demonstrated that TLR12, a previously uncharacterized TLR12, also recognizes TgPRF. Importantly, TLR12-deficient mice display markedly increased susceptibility to T. gondii infection relative to TLR11 KO animals and this defect is associated with decreased IFN-alpha production and reduced NK cell activity. Taken together, these studies establish a dual role for TLR11/TLR12 signaling in both innate recognition of T. gondii and host resistance to the parasite The recognition of T. gondii by human cells, which do not express TLR11 and TLR12 (because of the presence of a stop codon in the genes), is still, however, poorly understood. For this reason, as proposed in our last years report, we have initiated a new project aimed at characterizing the cytokine/chemokine response elicited in human peripheral blood myeloid cells upon exposure to Toxoplasma gondii tachyzoites and identifying the signaling pathways involved. When the response of elutriated monocytes and in vitro differentiated dendritic cells or macrophages was compared, the production of proinflammatory cytokines (e.g. TNF and p40 IL-12) and chemokines was largely restricted to monocytes and, in particular, to the CD14high subset stimulated following parasite exposure. Our results also indicate that while infection with live parasites is required for the induction of cytokine/chemokine responses by monocytes, similar responses are obtained regardless of the virulence properties of the particular T.gondii strain employed in the assay. As a potent inducer of Th1 effectors, T. gondii infection provides a unique model for studying the process of Th1 differentiation. In collaboration with Dr. John OShea group (NIAMS), we showed that activated CD4+ T lymphocytes rapidly acquire and maintain the expression of the transcriptional factor T-bet (a master regulator of Th1 cells), which in turn suppresses the expression of the transcriptional factor Bcl-6 required for the development of T follicular helper cells. In a separate collaboration with Drs. William Paul and Jeff Zhou, we employed mice that while lacking expression of endogenous T-bet, express as a transgene a T-bet reporter, to demonstrate that T-bet promotes the development of Th1 cells during T. gondii infection by repressing the expression of GATA-3 (a master regulator of Th2 cell differentiation). As summarized in previous reports, we have found that Th1 cells coexpress IFN-gamma and IL-10 in toxoplasma-infected mice. Since our initial description of IL-10+Th1 cells, CD4+ T lymphocytes with this phenotype has been demonstrated in numerous other Th1 inducing parasitic, bacterial and viral animal infection models, as well as in human PBMC cultures. However, while INF-gamma is a stable property of Th1 cells, IL-10 secretion is transient. The latter finding makes it particularly difficult to study the molecular mechanism(s) that regulate IL-10 expression in Th1 cells. In order to facilitate this task, the work completed this year, has focused on subdivision of in vivo generated Th1 cells into three populations: those that express IL-10, those poised to express the cytokine and those that are IL-10 negative. This was successfully achieved by employing two different types of IL-10 reporter mice. Comparative analysis of the three Th1 subpopulations revealed that IL-10 mRNA can be only detected in cells expressing IL-10, while evaluation of histone modifications by ChIP-seq demonstrated three distinct epigenetics configurations of the IL-10 gene specific for each Th1 phenotype. Since our earlier studies indicated that Stat1, but not Stat3, is required for IL-10+ expression in Th1 cells, we are currently investigating the role Stat1 plays in epigenetic modifications (e.g. induction of H3K4me3 (permissive) and suppression H3K27me3 (inhibitory) marks) associated with the fully activate Il10 gene. Immune responses triggered by pathogens are part of the physiological response of the host as a whole and as such may be under the influence of extrinsic controlling elements. For example, glucocorticoids (GS) a class of steroid hormones regulated by hypothalamic-pituitary-adrenal (HPA) axis are known to exert pleiotropic effects on immune cells. As reported last year, we showed that GC are induced during the late acute phase of T. gondii infection and that mice lacking GC receptor expression in T lymphocytes display rapid mortality despite efficient control of parasite growth and uncompromised expression of the regulatory cytokines (e.g. IL-10 and IL-27). To understand their increased susceptibility, we have now demonstrated that in mice that lack GC receptor in T cells Th1 cells display in vivo, but not in vitro, a hyperactive phenotype associated with increased immunopathology. Interestingly, while GC have been described to suppress IFN-γ, and augment IL-10, secretion by CD4 T lymphocytes, the lack of GC signaling in T. gondii-induced Th1 cells resulted in general overexpression of IFN-γ, TNF as well as IL-10. The latter result strongly suggests that, under the highly polarized Th1 conditions of toxoplasma infection, GC exert a type of general rheostat, rather than cytokine-targeted, regulation on Th1 effectors by increasing the threshold of Ag dose required for TCR activation. Importantly, we also showed that the CD4+ T cell response to T. gondii infection is itself required for increased GC levels. Taken together, our results demonstrate a novel regulatory circuit involving cross-talk between CD4 T lymphocytes and GC production. This pathway which acts independently of innate immune cell cytokine production plays an important role in balancing Th1 mediated pathogen clearance and tissue immunopathology.

Project Start
Project End
Budget Start
Budget End
Support Year
23
Fiscal Year
2012
Total Cost
$553,011
Indirect Cost
City
State
Country
Zip Code
Tosh, Kevin W; Mittereder, Lara; Bonne-Annee, Sandra et al. (2016) The IL-12 Response of Primary Human Dendritic Cells and Monocytes to Toxoplasma gondii Is Stimulated by Phagocytosis of Live Parasites Rather Than Host Cell Invasion. J Immunol 196:345-56
Sher, Alan; Tosh, Kevin; Jankovic, Dragana (2016) Innate recognition of Toxoplasma gondii in humans involves a mechanism distinct from that utilized by rodents. Cell Mol Immunol :
Yang, Xiang-Ping; Jiang, Kan; Hirahara, Kiyoshi et al. (2015) EZH2 is crucial for both differentiation of regulatory T cells and T effector cell expansion. Sci Rep 5:10643
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Jankovic, Dragana; Feng, Carl G (2015) CD4(+) T Cell Differentiation in Infection: Amendments to the Th1/Th2 Axiom. Front Immunol 6:198
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Giardino Torchia, Maria Letizia; Conze, Dietrich B; Jankovic, Dragana et al. (2013) Balance between NF-κB p100 and p52 regulates T cell costimulation dependence. J Immunol 190:549-55

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