An ex vivo model of Toxoplasma recrudescence reveals developmental plasticity of the bradyzoite stage.

IMPORTANCE: The classical depiction of the Toxoplasma lifecycle is bradyzoite excystation conversion to tachyzoites, cell lysis, and immune control, followed by the reestablishment of bradyzoites and cysts. In contrast, we show that tachyzoite growth slows independent of the host immune response at a predictable time point following excystation. Furthermore, we demonstrate a host cell-dependent pathway of continuous amplification of the cyst-forming bradyzoite population. The developmental plasticity of the excysted bradyzoites further underlines the critical role the cyst plays in the flexibility of the lifecycle of this ubiquitous parasite. This revised model of Toxoplasma recrudescence uncovers previously unknown complexity in the clinically important bradyzoite stage of the parasite, which opens the door to further study these novel developmental features of the Toxoplasma intermediate life cycle.

Group 1 metabotropic glutamate receptor expression defines a T cell memory population during chronic Toxoplasma infection that enhances IFN-gamma and perforin production in the CNS.

Within the brain, a pro-inflammatory response is essential to prevent clinical disease due to Toxoplasma gondii reactivation. Infection in the immunocompromised leads to lethal Toxoplasmic encephalitis while in the immunocompetent, there is persistent low-grade inflammation which is devoid of clinical symptoms. This signifies that there is a well-balanced and regulated inflammatory response to T. gondii in the brain. T cells are the dominant immune cells that prevent clinical disease, and this is mediated through the secretion of effector molecules such as perforins and IFN-γ. The presence of cognate antigen, the expression of survival cytokines, and the alteration of the epigenetic landscape drive the development of memory T cells. However, specific extrinsic signals that promote the formation and maintenance of memory T cells within tissue are poorly understood. During chronic infection, there is an increase in extracellular glutamate that, due to its function as an excitatory neurotransmitter, is normally tightly controlled in the CNS. Here we demonstrate that CD8+ T cells from the T. gondii-infected brain parenchyma are enriched for metabotropic glutamate receptors (mGluR's). Characterization studies determined that mGluR+ expression by CD8+ T cells defines a distinct memory population at the transcriptional and protein level. Finally, using receptor antagonists and agonists we demonstrate mGluR signaling is required for optimal CD8+ T cell production of the effector cytokine IFNγ. This work suggests that glutamate is an important environmental signal of inflammation that promotes T cell function. Understanding glutamate's influence on T cells in the brain can provide insights into the mechanisms that govern protective immunity against CNS-infiltrating pathogens and neuroinflammation.

A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes.

Astrocytes are vital support cells that ensure proper brain function. In brain disease, astrocytes reprogram into a reactive state that alters many of their cellular roles. A long-standing question in the field is whether downregulation of reactive astrocyte (RA) markers during resolution of inflammation is because these astrocytes revert back to a non-reactive state or die and are replaced. This has proven difficult to answer mainly because existing genetic tools cannot distinguish between healthy versus RAs. Here we describe the generation of an inducible genetic tool that can be used to specifically target and label a subset of RAs. Longitudinal analysis of an acute inflammation model using this tool revealed that the previously observed downregulation of RA markers after inflammation is likely due to changes in gene expression and not because of cell death. Our findings suggest that cellular changes associated with astrogliosis after acute inflammation are largely reversible.

Deconvolution of pro- and antiviral genomic responses in Zika virus-infected and bystander macrophages.

Genome-wide investigations of host-pathogen interactions are often limited by analyses of mixed populations of infected and uninfected cells, which lower sensitivity and accuracy. To overcome these obstacles and identify key mechanisms by which Zika virus (ZIKV) manipulates host responses, we developed a system that enables simultaneous characterization of genome-wide transcriptional and epigenetic changes in ZIKV-infected and neighboring uninfected primary human macrophages. We demonstrate that transcriptional responses in ZIKV-infected macrophages differed radically from those in uninfected neighbors and that studying the cell population as a whole produces misleading results. Notably, the uninfected population of macrophages exhibits the most rapid and extensive changes in gene expression, related to type I IFN signaling. In contrast, infected macrophages exhibit a delayed and attenuated transcriptional response distinguished by preferential expression of IFNB1 at late time points. Biochemical and genomic studies of infected macrophages indicate that ZIKV infection causes both a targeted defect in the type I IFN response due to degradation of STAT2 and reduces RNA polymerase II protein levels and DNA occupancy, particularly at genes required for macrophage identity. Simultaneous evaluation of transcriptomic and epigenetic features of infected and uninfected macrophages thereby reveals the coincident evolution of dominant proviral or antiviral mechanisms, respectively, that determine the outcome of ZIKV exposure.

A longitudinal systems immunologic investigation of acute Zika virus infection in an individual infected while traveling to Caracas, Venezuela.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus linked to devastating neurologic diseases. Immune responses to flaviviruses may be pathogenic or protective. Our understanding of human immune responses to ZIKV in vivo remains limited. Therefore, we performed a longitudinal molecular and phenotypic characterization of innate and adaptive immune responses during an acute ZIKV infection. We found that innate immune transcriptional and genomic responses were both cell type- and time-dependent. While interferon stimulated gene induction was common to all innate immune cells, the upregulation of important inflammatory cytokine genes was primarily limited to monocyte subsets. Additionally, genomic analysis revealed substantial chromatin remodeling at sites containing cell-type specific transcription factor binding motifs that may explain the observed changes in gene expression. In this dengue virus-experienced individual, adaptive immune responses were rapidly mobilized with T cell transcriptional activity and ZIKV neutralizing antibody responses peaking 6 days after the onset of symptoms. Collectively this study characterizes the development and resolution of an in vivo human immune response to acute ZIKV infection in an individual with pre-existing flavivirus immunity.

An IRF-3-, IRF-5-, and IRF-7-Independent Pathway of Dengue Viral Resistance Utilizes IRF-1 to Stimulate Type I and II Interferon Responses.

Interferon-regulatory factors (IRFs) are a family of transcription factors (TFs) that translate viral recognition into antiviral responses, including type I interferon (IFN) production. Dengue virus (DENV) and other clinically important flaviviruses are suppressed by type I IFN. While mice lacking the type I IFN receptor (Ifnar1-/-) succumb to DENV infection, we found that mice deficient in three transcription factors controlling type I IFN production (Irf3-/-Irf5-/-Irf7-/- triple knockout [TKO]) survive DENV challenge. DENV infection of TKO mice resulted in minimal type I IFN production but a robust type II IFN (IFN-γ) response. Using loss-of-function approaches for various molecules, we demonstrate that the IRF-3-, IRF-5-, IRF-7-independent pathway predominantly utilizes IFN-γ and, to a lesser degree, type I IFNs. This pathway signals via IRF-1 to stimulate interleukin-12 (IL-12) production and IFN-γ response. These results reveal a key antiviral role for IRF-1 by activating both type I and II IFN responses during DENV infection.

Mapping and Role of the CD8+ T Cell Response During Primary Zika Virus Infection in Mice.

CD8+ T cells may play a dual role in protection against and pathogenesis of flaviviruses, including Zika virus (ZIKV). We evaluated the CD8+ T cell response in ZIKV-infected LysMCre+IFNARfl/fl C57BL/6 (H-2b) mice lacking the type I interferon receptor in a subset of myeloid cells. In total, 26 and 15 CD8+ T cell-reactive peptides for ZIKV African (MR766) and Asian (FSS13025) lineage strains, respectively, were identified and validated. CD8+ T cells from infected mice were polyfunctional and mediated cytotoxicity. Adoptive transfer of ZIKV-immune CD8+ T cells reduced viral burdens, whereas their depletion led to higher tissue burdens, and CD8-/- mice displayed higher mortality with ZIKV infection. Collectively, these results demonstrate that CD8+ T cells protect against ZIKV infection. Further, this study provides a T cell competent mouse model for investigating ZIKV-specific T cell responses.

Twist1 induces CCL2 and recruits macrophages to promote angiogenesis.

The transcription factor Twist1 induces epithelial-mesenchymal transition and extracellular matrix degradation to promote tumor metastasis. Although Twist1 also plays a role in embryonic vascular development and tumor angiogenesis, the molecular mechanisms that underlie these processes are not as well understood. Here, we report a novel function for Twist1 in modifying the tumor microenvironment to promote progression. We found that expression of Twist1 in human mammary epithelial cells potently promoted angiogenesis. Surprisingly, Twist1 expression did not increase the secretion of the common proangiogenic factors VEGF and basic fibroblast growth factor but rather induced expression of the macrophage chemoattractant CCL2. Attenuation of endogenous Twist1 in vivo blocked macrophage recruitment and angiogenesis, whereas exogenous CCL2 rescued the ability of tumor cells lacking Twist1 to attract macrophages and promote angiogenesis. Macrophage recruitment also was essential for the ability of Twist1-expressing cells to elicit a strong angiogenic response. Together, our findings show that how Twist1 recruits stromal macrophages through CCL2 induction to promote angiogenesis and tumor progression. As Twist1 expression has been associated with poor survival in many human cancers, this finding suggests that anti-CCL2 therapy may offer a rational strategy to treat Twist1-positive metastatic cancers.