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PDF file - 618K, CCL2 is not required for Twist1-induced EMT.

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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. Cancer Res; 73(2); 662–71. ©2012 AACR.

PDF file - 66K, Expression of Twist1 in HMLE cells.

PDF file - 209K, Twist1 specifically induces the expression of CCL2, but not other angiogenic factors, such as VEGF and bFGF.

Current approaches to find therapeutic solutions to treat and prevent reactivation of toxoplasmosis have suffered from limited accessibility to the relevant Toxoplasma stages and a lack of accurate in vitro developmental models. The loss of developmental competency in vitro that is exacerbated during the generation of transgenic tachyzoites is also a major impediment to understanding the molecular basis of bradyzoite recrudescence, which is the central feature of reactivation. We have developed an innovative ex vivo model of bradyzoite recrudescence and applied this method to successfully modify Toxoplasma genes while avoiding the problems caused by continuous in vitro cell culture. We present four protocols required to engineer in vivo transgenic tissue cysts: 1) the reliable production of in vivo tissue cysts and excysted bradyzoites, 2) the use of fast-growing parasites from ex vivo bradyzoite infections to successfully generate transgenic tissue cysts in mice, 3) the cloning of transgenic bradyzoites via single cyst infections, and finally, 4) the long term cold storage and recovery of transgenic tissue cysts in brain tissue homogenates. We demonstrated these protocols by knocking out the Toxoplasma HXGPRT gene and the gene encoding the ApiAP2 transcription factor, AP2IX-9 in tissue cysts from mice. Unexpectedly, the knockout of the AP2IX-9 gene in the Type II ME49EW strain eliminated one of the three developmental pathways initiated by the bradyzoite; host-dependent bradyzoite to bradyzoite replication.

Efforts to develop therapies against Toxoplasma reactivation have been hampered by the lack of an in vitro model of bradyzoite recrudescence. To overcome this issue, we established a new ex vivo model of bradyzoite recrudescence using bradyzoites from an unadapted Type II ME49 strain isolated from murine brain tissue to infect human foreskin fibroblasts and neonatal murine astrocytes. Bradyzoite infection of both host cell types produced two sequential tachyzoite growth stages that appeared similar to previous sporozoite in vitro infections; a fast-growing stage was followed by spontaneous formation of a slower-growing stage. In astrocytes, but not in fibroblasts, there was evidence of second bradyzoite to bradyzoite recrudescent pathway that occurred simultaneously with the bradyzoite to tachyzoite pathway. Thus, the host cell environment strongly influenced the pathway of bradyzoite recrudescence. Infections of mice with either the fast-growing or slow-growing recrudescent tachyzoites showed progressive decreases in brain cyst formation that could be fully recovered using serial tissue cyst passage demonstrating the loss of developmental capacity was reversible. By contrast, poor tissue cyst formation of laboratory-adapted tachyzoites was not reversible by these approaches indicating developmental incompetence was permanent in these strains. In order to develop methods to distinguish strain developmental competency, we identified Toxoplasma genes highly expressed in ME49EW in vivo tissue cysts and developed a qPCR approach that differentiates immature from mature bradyzoites. In summary, the results presented describe a new ex vivo bradyzoite recrudescence model that fully captures the reported growth and developmental processes observed during toxoplasmosis reactivation in vivo opening the door to the further study of these important features of the Toxoplasma intermediate life cycle.

Summary 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 + IFNAR fl/fl C57BL/6 (H-2 b ) 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.

Several Zika virus (ZIKV) vaccines designed to elicit protective antibody (Ab) responses are currently under rapid development, but the underlying mechanisms that control the magnitude and quality of the Ab response remain unclear. Here, we investigated the CD4+ T cell response to primary intravenous and intravaginal infection with ZIKV. Using the LysMCre+Ifnar1fl/fl (myeloid type I IFN receptor-deficient) C57BL/6 mouse models, we identified six I-Ab-restricted ZIKV epitopes that stimulated CD4+ T cells with a predominantly cytotoxic Th1 phenotype in mice primed with ZIKV. Intravenous and intravaginal infection with ZIKV effectively induced follicular helper and regulatory CD4+ T cells. Treatment of mice with a CD4+ T cell-depleting Ab reduced the plasma cell, germinal center B cell, and IgG responses to ZIKV without affecting the CD8+ T cell response. CD4+ T cells were required to protect mice from a lethal dose of ZIKV after infection intravaginally, but not intravenously. However, adoptive transfer and peptide immunization experiments showed a role for memory CD4+ T cells in ZIKV clearance in mice challenged intravenously. These results demonstrate that CD4+ T cells are required mainly for the generation of a ZIKV-specific humoral response but not for an efficient CD8+ T cell response. Thus, CD4+ T cells could be important mediators of protection against ZIKV, depending on the infection or vaccination context.

Significance Interpretation of genome-wide investigations of host–pathogen interactions are often obscured by analyses of mixed populations of infected and uninfected cells. Thus, we developed a system whereby we simultaneously characterize and compare genome-wide transcriptional and epigenetic changes in pure populations of virally infected and neighboring uninfected cells to identify viral-regulated host responses. Using patient-derived unmodified Zika viruses (ZIKV) infecting primary human macrophages, we reveal that ZIKV suppresses host transcription by multiple mechanisms. ZIKV infection causes both targeted suppression of type I interferon responses and general suppression by reducing RNA polymerase II protein levels and DNA occupancy. Simultaneous evaluation of transcriptomic and epigenetic features of infected and uninfected cells provides a powerful method for identifying coincident evolution of dominant proviral or antiviral mechanisms., 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.

Several Zika virus (ZIKV) vaccines designed to elicit protective antibody (Ab) responses are currently under rapid development, but the underlying mechanisms that control the magnitude and quality of the Ab response remain unclear. Here, we investigated the CD4+ T cell response to primary intravenous and intravaginal infection with ZIKV. Using the LysMCre+Ifnar1fl/fl (myeloid type I IFN receptor-deficient) C57BL/6 mouse models, we identified six I-Ab-restricted ZIKV epitopes that stimulated CD4+ T cells with a predominantly cytotoxic Th1 phenotype in mice primed with ZIKV. Intravenous and intravaginal infection with ZIKV effectively induced follicular helper and regulatory CD4+ T cells. Treatment of mice with a CD4+ T cell-depleting Ab reduced the plasma cell, germinal center B cell, and IgG responses to ZIKV without affecting the CD8+ T cell response. CD4+ T cells were required to protect mice from a lethal dose of ZIKV after infection intravaginally, but not intravenously. However, adoptive transfer and peptide immunization experiments showed a role for memory CD4+ T cells in ZIKV clearance in mice challenged intravenously. These results demonstrate that CD4+ T cells are required mainly for the generation of a ZIKV-specific humoral response but not for an efficient CD8+ T cell response. Thus, CD4+ T cells could be important mediators of protection against ZIKV, depending on the infection or vaccination context., Author summary Several vaccine candidates are currently under development for Zika virus (ZIKV) infection, which causes life-threatening neurologic diseases, including congenital Zika syndrome in neonates and Guillain-Barré syndrome in adults. However, the mechanisms by which the adaptive immune system contributes to protection against ZIKV have not been fully investigated. In this report, we used mouse models of systemic and genital mucosal infection with ZIKV to evaluate the role of CD4+ T cells in regulating antiviral humoral and CD8+ T cell responses and in controlling ZIKV infection. We first defined the antigen specificity and phenotype of the CD4+ T cell response to ZIKV and then demonstrated that CD4+ T cells are required for inducing humoral, but not CD8+ T cell, responses in ZIKV-primed mice. CD4+ T cells were also required for local control of ZIKV burden in mice infected intravaginally. Although CD4+ T cells are not necessary for the control of primary ZIKV infection via the intravenous route, CD4+ T cells can confer protection against lethal intravaginal ZIKV challenge. Our results support ZIKV vaccine strategies that induce a protective CD4+ T cell response to ZIKV.

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., Author summary Zika virus (ZIKV) is an emerging flaviviral infection that causes significant clinical disease. It is estimated that approximately one half of the world’s population is at risk for ZIKV infection. There are only a limited number of studies describing the human immune response to ZIKV infection. Carlin et al. combined conventional and genomic approaches to longitudinally analyze the innate and adaptive immune responses to acute ZIKV infection and its resolution in a person who was infected while traveling in Venezuela during the 2016 ZIKV epidemic year. Genome-wide sequencing in individual cell types revealed that although many populations respond to interferon stimulation, only specific cell populations within peripheral blood mononuclear cells upregulate important inflammatory cytokine gene expression. Additionally, analysis of open chromatin using ATAC-seq suggests that chromatin remodeling at sites containing cell-type specific transcription factor binding motifs may help us understand changes in gene expression. Consistent with previous reports, this individual with prior exposure to dengue virus (DENV), rapidly developed neutralizing anti-ZIKV responses that were cross-reactive with multiple DENV serotypes. Collectively this study combines traditional and genomic approaches to characterize the cell-type specific development of an in vivo human immune response to acute ZIKV infection.

CD8

Summary: 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. : Carlin et al. identify a non-canonical IRF-3-, IRF-5-, and IRF-7-independent antiviral defense mechanism that mediates protection against severe dengue disease. This alternative pathway utilizes IRF-1, predominantly via IL-12/IFN-γ, enabling survival in the context of reduced type I IFN responses. Keywords: dengue, interferon, IFNs, IRF-1, IRFs, mouse models, macrophages, IL-12, innate immunity, flavivirus

CD8+ T cells may play a dual role in both protection against and pathogenesis of Flaviviruses, including Zika virus (ZIKV). However, virtually nothing is currently known about the role CD8+ T cells play during ZIKV infection in humans and animal models. The goal of our study is to investigate the role of CD8+ T cells during primary ZIKV infection in mice. Using LysMCre+IFNARfl/fl C57BL/6 (H-2b) mice that lack type I IFN receptor in a subset of myeloid cells, we characterized a new mouse model susceptible to infection with both East African lineage (MR766) and Asian lineage (FSS13025) ZIKV strains. A screening of 244 ZIKV epitopes predicted to bind H-2b class I molecules, defined a specific map of 26 and 15 peptides for MR766 and FSS13025 ZIKV strains, respectively. Intracellular cytokine staining confirmed the identity of these epitopes and demonstrated induction of polyfunctional ZIKV-specific CD8+ T cells. Furthermore, adoptive transfer of ZIKV-immune CD8+ T cells reduced viral burdens, whereas depletion of CD8+ T cells led to higher tissue burdens and mortality was increased in ZIKV-infected CD8−/− mice compared to Wild-type. These results demonstrate that CD8+ T cells are important mediators of protection during primary ZIKV infection and provide a H-2b mouse model for investigating ZIKV-specific CD8+ T cell responses. Based on these findings, studies are in progress towards defining the role of resident memory CD8+ T cells in protection against ZIKV infection using our recently developed mouse models of ZIKV systemic infection and sexual transmission.

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. Cancer Res; 73(2); 662–71. ©2012 AACR.