Both Functional LTβ Receptor and TNF Receptor 2 Are Required for the Development of Experimental Cerebral Malaria

Background

TNF-related lymphotoxin α (LTα) is essential for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The pathway involved has been attributed to TNFR2. Here we show a second arm of LTα-signaling essential for ECM development through LTβ-R, receptor of LTα1β2 heterotrimer.

Methodology/Principal Findings

LTβR deficient mice did not develop the neurological signs seen in PbA induced ECM but died at three weeks with high parasitaemia and severe anemia like LTαβ deficient mice. Resistance of LTαβ or LTβR deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and angiography, associated with lack of microvascular obstruction, while wild-type mice developed distinct microvascular pathology. Recruitment and activation of perforin⁺ CD8⁺ T cells, and their ICAM-1 expression were clearly attenuated in the brain of resistant mice. An essential contribution of LIGHT, another LTβR ligand, could be excluded, as LIGHT deficient mice rapidly succumbed to ECM.

Conclusions/Significance

LTβR expressed on radioresistant resident stromal, probably endothelial cells, rather than hematopoietic cells, are essential for the development of ECM, as assessed by hematopoietic reconstitution experiment. Therefore, the data suggest that both functional LTβR and TNFR2 signaling are required and non-redundant for the development of microvascular pathology resulting in fatal ECM.     

Experimental Cerebral Malaria Pathogenesis—Hemodynamics at the Blood Brain Barrier

Cerebral malaria claims the lives of over 600,000 African children every year. To better understand the pathogenesis of this devastating disease, we compared the cellular dynamics in the cortical microvasculature between two infection models, Plasmodium berghei ANKA (PbA) infected CBA/CaJ mice, which develop experimental cerebral malaria (ECM), and P. yoelii 17XL (PyXL) infected mice, which succumb to malarial hyperparasitemia without neurological impairment. Using a combination of intravital imaging and flow cytometry, we show that significantly more CD8+ T cells, neutrophils, and macrophages are recruited to postcapillary venules during ECM compared to hyperparasitemia. ECM correlated with ICAM-1 upregulation on macrophages, while vascular endothelia upregulated ICAM-1 during ECM and hyperparasitemia. The arrest of large numbers of leukocytes in postcapillary and larger venules caused microrheological alterations that significantly restricted the venous blood flow. Treatment with FTY720, which inhibits vascular leakage, neurological signs, and death from ECM, prevented the recruitment of a subpopulation of CD45^hi CD8+ T cells, ICAM-1+ macrophages, and neutrophils to postcapillary venules. FTY720 had no effect on the ECM-associated expression of the pattern recognition receptor CD14 in postcapillary venules suggesting that endothelial activation is insufficient to cause vascular pathology. Expression of the endothelial tight junction proteins claudin-5, occludin, and ZO-1 in the cerebral cortex and cerebellum of PbA-infected mice with ECM was unaltered compared to FTY720-treated PbA-infected mice or PyXL-infected mice with hyperparasitemia. Thus, blood brain barrier opening does not involve endothelial injury and is likely reversible, consistent with the rapid recovery of many patients with CM. We conclude that the ECM-associated recruitment of large numbers of activated leukocytes, in particular CD8+ T cells and ICAM+ macrophages, causes a severe restriction in the venous blood efflux from the brain, which exacerbates the vasogenic edema and increases the intracranial pressure. Thus, death from ECM could potentially occur as a consequence of intracranial hypertension.     

Phosphatidylinositol 3-Kinase Class II α-Isoform PI3K-C2α Is Required for Transforming Growth Factor β-induced Smad Signaling in Endothelial Cells

We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P₁ and thereby their signaling on endosomes. TGFβ, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFβ1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFβ-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFβ receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFβ receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFβ receptor internalization and Smad2/3 phosphorylation. TGFβ1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFβ1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFβ receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFβ.     

The Class II Phosphatidylinositol 3 kinase C2β Is Required for the Activation of the K+ Channel KCa3.1 and CD4 T-Cells

The Ca²⁺-activated K⁺ channel KCa3.1 is required for Ca²⁺ influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2β (PI3K-C2β) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2β by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2β siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2β in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca²⁺ influx, whereas silencing of PI3K-C2β inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2β colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.     

A TCRβ Repertoire Signature Can Predict Experimental Cerebral Malaria

Cerebral Malaria (CM) is associated with a pathogenic T cell response. Mice infected by P. berghei ANKA clone 1.49 (PbA) developing CM (CM⁺) present an altered PBL TCR repertoire, partly due to recurrently expanded T cell clones, as compared to non-infected and CM^- infected mice. To analyse the relationship between repertoire alteration and CM, we performed a kinetic analysis of the TRBV repertoire during the course of the infection until CM-related death in PbA-infected mice. The repertoires of PBL, splenocytes and brain lymphocytes were compared between infected and non-infected mice using a high-throughput CDR3 spectratyping method. We observed a modification of the whole TCR repertoire in the spleen and blood of infected mice, from the fifth and the sixth day post-infection, respectively, while only three TRBV were significantly perturbed in the brain of infected mice. Using multivariate analysis and statistical modelling, we identified a unique TCRβ signature discriminating CM⁺ from CTR mice, enriched during the course of the infection in the spleen and the blood and predicting CM onset. These results highlight a dynamic modification and compartmentalization of the TCR diversity during the course of PbA infection, and provide a novel method to identify disease-associated TCRβ signature as diagnostic and prognostic biomarkers.     

Phospholipase Cγ2 Is Required for Luminal Expansion of the Epididymal Duct during Postnatal Development in Mice

Phospholipase Cγ2 (PLCγ2)-deficient mice exhibit misconnections of blood and lymphatic vessels, and male infertility. However, the cell type responsible for vascular partitioning and the mechanism for male infertility remain unknown. Accordingly, we generated a mouse line that conditionally expresses endogenous Plcg2 in a Cre/loxP recombination-dependent manner, and found that Tie2-Cre- or Pf4-Cre-driven reactivation of Plcg2 rescues PLCγ2-deficient mice from the vascular phenotype. By contrast, male mice rescued from the vascular phenotype exhibited epididymal sperm granulomas. As judged from immunostaining, PLCγ2 was expressed in clear cells in the epididymis. PLCγ2 deficiency did not compromise differentiation of epididymal epithelial cells, including clear cells, and tube formation at postnatal week 2. However, luminal expansion of the epididymal duct was impaired during the prepubertal period, regardless of epithelial cell polarity and tube architecture. These results suggest that PLCγ2-deficient clear cells cause impaired luminal expansion, stenosis of the epididymal duct, attenuation of luminal flow, and subsequent sperm granulomas. Clear cell-mediated luminal expansion is also supported by the observation that PLCγ2-deficient males were rescued from infertility by epididymal epithelium-specific reactivation of Plcg2, although the edematous and hemorrhagic phenotype associated with PLCγ2 deficiency also caused spontaneous epididymal sperm granulomas in aging males. Collectively, our findings demonstrate that PLCγ2 in clear cells plays an essential role in luminal expansion of the epididymis during the prepubertal period in mice, and reveal an unexpected link between PLCγ2, clear cells, and epididymal development.     

Insulin Secretion Induced by Glucose-dependent Insulinotropic Polypeptide Requires Phosphatidylinositol 3-Kinase γ in Rodent and Human β-Cells

PI3Kγ, a G-protein-coupled type 1B phosphoinositol 3-kinase, exhibits a basal glucose-independent activity in β-cells and can be activated by the glucose-dependent insulinotropic polypeptide (GIP). We therefore investigated the role of the PI3Kγ catalytic subunit (p110γ) in insulin secretion and β-cell exocytosis stimulated by GIP. We inhibited p110γ with AS604850 (1 μmol/liter) or knocked it down using an shRNA adenovirus or siRNA duplex in mouse and human islets and β-cells. Inhibition of PI3Kγ blunted the exocytotic and insulinotropic response to GIP receptor activation, whereas responses to the glucagon-like peptide-1 or the glucagon-like peptide-1 receptor agonist exendin-4 were unchanged. Downstream, we find that GIP, much like glucose stimulation, activates the small GTPase protein Rac1 to induce actin remodeling. Inhibition of PI3Kγ blocked these effects of GIP. Although exendin-4 could also stimulate actin remodeling, this was not prevented by p110γ inhibition. Finally, forced actin depolymerization with latrunculin B restored the exocytotic and secretory responses to GIP during PI3Kγ inhibition, demonstrating that the loss of GIP-induced actin depolymerization was indeed limiting insulin exocytosis.     

LRP1 Regulates Architecture of the Vascular Wall by Controlling PDGFRβ-Dependent Phosphatidylinositol 3-Kinase Activation

Background

Low density lipoprotein receptor-related protein 1 (LRP1) protects against atherosclerosis by regulating the activation of platelet-derived growth factor receptor β (PDGFRβ) in vascular smooth muscle cells (SMCs). Activated PDGFRβ undergoes tyrosine phosphorylation and subsequently interacts with various signaling molecules, including phosphatidylinositol 3-kinase (PI3K), which binds to the phosphorylated tyrosine 739/750 residues in mice, and thus regulates actin polymerization and cell movement.

Methods and Principal Findings

In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP1−/−) SMCs. Marfan syndrome-like phenotypes such as tortuous aortas, disrupted elastic layers and abnormally activated transforming growth factor β (TGFβ) signaling are present in smooth muscle-specific LRP1 knockout (smLRP1−/−) mice. To investigate the role of LRP1-regulated PI3K activation by PDGFRβ in atherogenesis, we generated a strain of smLRP1−/− mice in which tyrosine 739/750 of the PDGFRβ had been mutated to phenylalanines (PDGFRβ F2/F2). Spontaneous atherosclerosis was significantly reduced in the absence of hypercholesterolemia in these mice compared to smLRP1−/− animals that express wild type PDGFR. Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFβ signaling, as indicated by high levels of nuclear phospho-Smad2.

Conclusions and Significance

Our data suggest that LRP1 regulates actin organization and cell migration by controlling PDGFRβ-dependent activation of PI3K. TGFβ activation alone is not sufficient for the expression of the Marfan-like vascular phenotype. Thus, regulation of PI3 Kinase by PDGFRβ is essential for maintaining vascular integrity, and for the prevention of atherosclerosis as well as Marfan syndrome.     

Topoisomerase 3α Is Required for Decatenation and Segregation of Human mtDNA

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β-Catenin Is Required for the Tumorigenic Behavior of Triple-Negative Breast Cancer Cells

Our previous data illustrated that activation of the canonical Wnt signaling pathway was enriched in triple-negative breast cancer and associated with reduced overall survival in all patients. To determine whether Wnt signaling may be a promising therapeutic target for triple-negative breast cancer, we investigated whether β-catenin was necessary for tumorigenic behaviors in vivo and in vitro. β-catenin expression level was significantly reduced in two human triple-negative breast cancer cell lines, MDA-MB-231 and HCC38, using lentiviral delivery of β-catenin-specific small hairpin RNAs (shRNAs). Upon implantation of the cells in the mammary fat pad of immunocompromised mice, we found that β-catenin shRNA HCC38 cells formed markedly smaller tumors than control cells and grew much more slowly. In in vitro assays, β-catenin silencing significantly reduced the percentage of Aldefluor-positive cells, a read-out of the stem-like cell population, as well as the expression of stem cell-related target genes including Bmi-1 and c-Myc. β-catenin-knockdown cells were also significantly impaired in their ability to migrate in wound-filling assays and form anchorage-independent colonies in soft agar. β-catenin-knockdown cells were more sensitive to chemotherapeutic agents doxorubicin and cisplatin. Collectively, these data suggest that β-catenin is required for triple-negative breast cancer development by controlling numerous tumor-associated properties, such as migration, stemness, anchorage-independent growth and chemosensitivity.     

Class IA Phosphatidylinositol 3-Kinase p110α Regulates Phagosome Maturation

Of the various phosphatidylinositol 3- kinases (PI3Ks), only the class III enzyme Vps34 has been shown to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in class IA PI3K p110α, we discovered that this PI3K isoform is required for vacuole maturation to progress beyond acquisition of Rab7 leading to delivery of lysosomal markers. Bead phagosomes from THP-1 cells acquired p110α and contained PI3P and PI(3,4,5)P3; however, p110α and PI(3,4,5)P3 levels in phagosomes from p110α knockdown cells were decreased. Phagosomes from p110α knock down cells showed normal acquisition of both Rab5 and EEA-1, but were markedly deficient in the lysosomal markers LAMP-1 and LAMP-2, and the lysosomal hydrolase, β-galactosidase. Phagosomes from p110α deficient cells also displayed impaired fusion with Texas Red dextran-loaded lysosomes. Despite lacking lysosomal components, phagosomes from p110α deficient cells recruited normal levels of Rab7, Rab-interacting lysosomal protein (RILP) and homotypic vacuole fusion and protein sorting (HOPs) components Vps41 and Vps16. The latter observations demonstrated that phagosomal Rab7 was active and capable of recruiting effectors involved in membrane fusion. Nevertheless, active Rab7 was not sufficient to bring about the delivery of lysosomal proteins to the maturing vacuole, which is shown for the first time to be dependent on a class I PI3K.     

Gα and Gβ Proteins Regulate the Cyclic AMP Pathway That Is Required for Development and Pathogenicity of the Phytopathogen Mycosphaerella graminicola

We identified and functionally characterized genes encoding three Gα proteins and one Gβ protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian Gα_i and Gα_s families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB), MgGpa1 mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation. MgGpa3 mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of MgGpb1 mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of MgGpb1 and MgGpa3 mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in MgGpa1 mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of MgGpa1, MgGpa3, and MgGpb1 are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by MgGpa3 and MgGpb1 regulate the cAMP pathway that is required for development and pathogenicity in M. graminicola.Signal transduction pathways are important for sensing and responding to different environmental stimuli in both lower and higher eukaryotes. The highly conserved heterotrimeric guanine nucleotide-binding proteins (G proteins) belong to a family of regulatory proteins that are crucial for the transduction of signals, which are perceived by a distinct family of cell surface receptors (4). Heterotrimeric G proteins contain three subunits (α, β, and γ) that are linked in the inactive state. Activation of a Gα subunit by a transmembrane receptor leads to exchange of bound GDP with GTP on the Gα subunit, resulting in dissociation of the Gα and the Gβγ dimeric subunits, which can now interact with downstream effectors that subsequently generate changes in cellular responses (for a review, see reference 10).Filamentous fungi have one Gβ- and usually three Gα-encoding genes that belong to three major groups. Encoded proteins in groups I and III are related to the mammalian Gα_i and Gα_s families, respectively, but group II fungal Gα proteins have no mammalian counterpart (1, 4, 14, 22, 33, 53). Interestingly, the corn smut fungus Ustilago maydis contains a unique fourth Gα-encoding gene, and Saccharomyces cerevisiae contains only two Gα proteins (10, 57). Irrespective of the observed numerical variation, Gα proteins regulate a variety of cellular and developmental responses (4). For plant-pathogenic fungi, Gβ-encoding genes have been characterized functionally (9, 14, 22, 27, 31, 48, 52). Apart from the fact that individual Gα-encoding genes and the Gβ-encoding gene have been demonstrated to regulate growth, reproduction, and virulence, comparative functional characterization of all Gα-encoding genes has been reported only for a few plant-pathogenic fungi, including Magnaporthe grisea, Cryphonectria parasitica, and U. maydis (5, 41, 57).Mycosphaerella graminicola (anamorph Septoria tritici) causes septoria tritici blotch disease in bread and durum wheat in areas with high rainfall during the growing season, particularly in Western Europe, where it is considered to be the most important wheat disease (30). It is a ubiquitous phytopathogen with a lifestyle completely different from that of the aforementioned plant-pathogenic fungi. It is a dimorphic pathogen, and therefore the transition from a yeast-like to a filamentous form is important for initiation of infection (45). M. graminicola does not form appressoria but penetrates the leaves through stomata without forming specific infection structures. Furthermore, as a hemibiotroph, it has a biotrophic phase of about 10 days that is followed by a rapid switch to necrotrophy. The necrotic foliar lesions bear anamorphic and teleomorphic fructifications. M. graminicola is the model fungus for the Mycosphaerellaceae and even for the order Dothideales, an extremely large and diverse class of fungi with over 1,000 named species, including major plant pathogens such as the banana leaf streak fungus Mycosphaerella fijiensis (12, 21). Large expressed sequence tag (EST) libraries and the recently released genome sequence have been instrumental for the identification and characterization of genes involved in the development and pathogenicity of M. graminicola (http://genome.jgi-psf.org/Mycgr3/Mycgr3.home.html). Recently, we reported that genes encoding mitogen-activated protein kinases (MAPKs) (MgFus3, MgSlt2, and MgHog1) and the catalytic (MgTpk2) and regulatory (MgBcy1) subunits of protein kinase A (PKA) are essential pathogenicity factors and regulate specific steps during the infection process (8, 43-45). To extend our knowledge about the role of G proteins in the development and pathogenicity of M. graminicola, we functionally analyzed three Gα-encoding genes and one Gβ-encoding gene of M. graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Our results show the requirement of MgGpa1, MgGpa3, and MgGpb1 for pathogenicity, whereas the latter also negatively regulates cell fusion and anastomosis. Among the G protein-encoding genes characterized in this study, MgGpa3 and MgGpb1 positively regulate the cyclic AMP (cAMP) pathway. MgGpa1 seems to be dispensable for cAMP regulation, whereas MgGpa2 appears to be redundant, for none of the assays rendered altered phenotypes. Our results open new perspectives for studying the regulatory machinery of the cAMP pathway in M. graminicola and other plant-pathogenic fungi.