Emma Nye

Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8α+ dendritic cells

In mouse, a subset of dendritic cells (DCs) known as CD8α+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However, translation into clinical protocols has been hampered by the failure to identify CD8α+ DCs in humans. Here, we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8α+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8α+ DCs, human DNGR-1+ BDCA3hi DCs express Necl2, CD207, BATF3, IRF8, and TLR3, but not CD11b, IRF4, TLR7, or (unlike CD8α+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8, but not of TLR7, and produce interleukin (IL)-12 when given innate and T cell–derived signals. Notably, DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy.

Binding of Ras to Phosphoinositide 3-Kinase p110α Is Required for Ras- Driven Tumorigenesis in Mice

Ras proteins signal through direct interaction with a number of effector enzymes, including type I phosphoinositide (PI) 3-kinases. Although the ability of Ras to control PI 3-kinase has been well established in manipulated cell culture models, evidence for a role of the interaction of endogenous Ras with PI 3-kinase in normal and malignant cell growth in vivo has been lacking. Here we generate mice with mutations in the Pi3kca gene encoding the catalytic p110alpha isoform that block its interaction with Ras. Cells from these mice show proliferative defects and selective disruption of signaling from growth factors to PI 3-kinase. The mice display defective development of the lymphatic vasculature, resulting in perinatal appearance of chylous ascites. Most importantly, they are highly resistant to endogenous Ras oncogene-induced tumorigenesis. The interaction of Ras with p110alpha is thus required in vivo for certain normal growth factor signaling and for Ras-driven tumor formation.

Renal Cyst Formation in Fh1-Deficient Mice Is Independent of the Hif/Phd Pathway: Roles for Fumarate in KEAP1 Succination and Nrf2 Signaling

Summary The Krebs cycle enzyme fumarate hydratase (FH) is a human tumor suppressor whose inactivation is associated with the development of leiomyomata, renal cysts, and tumors. It has been proposed that activation of hypoxia inducible factor (HIF) by fumarate-mediated inhibition of HIF prolyl hydroxylases drives oncogenesis. Using a mouse model, we provide genetic evidence that Fh1-associated cyst formation is Hif independent, as is striking upregulation of antioxidant signaling pathways revealed by gene expression profiling. Mechanistic analysis revealed that fumarate modifies cysteine residues within the Kelch-like ECH-associated protein 1 (KEAP1), abrogating its ability to repress the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response pathway, suggesting a role for Nrf2 dysregulation in FH-associated cysts and tumors.

Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation.

Neutrophil recruitment is an important early step in controlling tissue infections or injury. Here, we report that this influx depends on both tissue-resident mast cells and macrophages. Mice with mast cell deficiency recruit reduced numbers of neutrophils in the first few hours of intraperitoneal lipopolysaccharide (LPS) stimulation. Conversely, in mice with clodronate-ablated macrophages, neutrophils extravasate, but have limited ability to reach the peritoneal fluid. Tissue macrophages synthesize neutrophil chemoattractants CXCL1/CXCL2 (CXC chemokine ligands 1/2) in response to LPS. Mast cells also produce these chemokines of which a proportion are preformed in granules. Release of the granules and new CXCL1/CXCL2 synthesis is Toll-like receptor 4-dependent. Both in vivo studies with blocking monoclonal antibodies and in vitro chemotaxis experiments show the neutrophil response to mast cells and macrophages to be CXCL1/CXCL2-dependent. The data are in keeping with the model that mast cells, optimally positioned in close proximity to the vasculature, initiate an early phase of neutrophil recruitment by releasing the chemoattractants CXCL1/CXCL2. Having arrived within the stimulated tissue, neutrophils penetrate further in a macrophage-dependent manner. Therefore, we demonstrate a positive role for mast cells in tissue inflammation and define how this comes about with contribution from a second tissue cell, the macrophage.

Endothelial cell O-glycan deficiency causes blood/lymphatic misconnections and consequent fatty liver disease in mice

Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1-derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn(-/-) mice). EHC T-syn(-/-) mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn(-/-) mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn(-/-) lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn(-/-) defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn(-/-) mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn(-/-) pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.

The GATA2 transcriptional network is requisite for RAS oncogene-driven non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer deaths worldwide; nearly half contain mutations in the receptor tyrosine kinase/RAS pathway. Here we show that RAS-pathway mutant NSCLC cells depend on the transcription factor GATA2. Loss of GATA2 reduced the viability of NSCLC cells with RAS-pathway mutations, whereas wild-type cells were unaffected. Integrated gene expression and genome occupancy analyses revealed GATA2 regulation of the proteasome, and IL-1-signaling, and Rho-signaling pathways. These pathways were functionally significant, as reactivation rescued viability after GATA2 depletion. In a Kras-driven NSCLC mouse model, Gata2 loss dramatically reduced tumor development. Furthermore, Gata2 deletion in established Kras mutant tumors induced striking regression. Although GATA2 itself is likely undruggable, combined suppression of GATA2-regulated pathways with clinically approved inhibitors caused marked tumor clearance. Discovery of the nononcogene addiction of KRAS mutant lung cancers to GATA2 presents a network of druggable pathways for therapeutic exploitation.

Murine but not human mesenchymal stem cells generate osteosarcoma-like lesions in the lung.

Murine mesenchymal stem cells are capable of differentiation into multiple cell types both in vitro and in vivo and may be good candidates to use as cell therapy for diseased or damaged organs. We have previously reported a method of enriching a population of murine MSCs that demonstrated a diverse differentiation potential both in vitro and in vivo. In this study, we show that this enriched population of murine mesenchymal stem cells embolize within lung capillaries following systemic injection and then rapidly expand within, and invade into, the lung parenchyma, forming tumor nodules. These lesions rarely contain cells bearing the immunohistochemical characteristics of lung epithelium, but they do show the characteristics of immature bone and cartilage that resembles exuberant fracture callus or well‐differentiated osteosarcoma. Our findings indicate that murine mesenchymal stem cells can behave in a manner similar to tumor cells, with dysregulated growth and aberrant differentiation within the alveolar microenvironment after four passages. We demonstrate that unlike human MSCs, MSCs from different mouse strains can acquire chromosomal abnormalities after only a few in vitro passages. Moreover, other parameters, such as mouse strain used, might also play a role in the induction of these tumors. These findings might be clinically relevant for future stem cell therapy studies.

JNK signalling modulates intestinal homeostasis and tumourigenesis in mice

Wnt signalling is a crucial signalling pathway controlling intestinal homeostasis and cancer. We show here that the JNK MAP kinase pathway and one of its most important substrates, the AP‐1 transcription factor c‐Jun, modulates Wnt signalling strength in the intestine. Transgenic gut‐specific augmentation of JNK signalling stimulated progenitor cell proliferation and migration, resulting in increased villus length. In the crypt, c‐Jun protein was highly expressed in progenitor cells and the absence of c‐Jun resulted in decreased proliferation and villus length. In addition to several known c‐Jun/AP‐1 target genes, expression of Wnt target genes Axin2 and Lgr5 were stimulated by JNK activation, suggesting a cross talk of JNK to Wnt signalling. Expression of the Wnt pathway component TCF4 was controlled by JNK activity, and chromatin immunoprecipitation and reporter assays identified tcf4 as a direct c‐Jun target gene. Consequently, increased JNK activity accelerated tumourigenesis in a model of colorectal carcinogenesis. As c‐jun is a direct target of the TCF4/β‐catenin complex, the control of tcf4 expression by JNK/c‐Jun leads to a positive feedback loop that connects JNK and Wnt signalling. This mechanism regulates the physiological function of progenitor cells and oncogenic transformation.

Bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fibrosis.

Many common diseases of the gas exchange surface of the lung have no specific treatment but cause serious morbidity and mortality. Idiopathic Pulmonary Fibrosis (IPF) is characterized by alveolar epithelial cell injury, interstitial inflammation, fibroblast proliferation and collagen accumulation within the lung parenchyma. Keratinocyte Growth Factor (KGF, also known as FGF-7) is a critical mediator of pulmonary epithelial repair through stimulation of epithelial cell proliferation. During repair, the lung not only uses resident cells after injury but also recruits circulating bone marrow-derived cells (BMDC). Several groups have used Mesenchymal Stromal Cells (MSCs) as therapeutic vectors, but little is known about the potential of Hematopoietic Stem cells (HSCs). Using an inducible lentiviral vector (Tet-On) expressing KGF, we were able to efficiently transduce both MSCs and HSCs, and demonstrated that KGF expression is induced in a regulated manner both in vitro and in vivo. We used the in vivo bleomycin-induced lung fibrosis model to assess the potential therapeutic effect of MSCs and HSCs. While both populations reduced the collagen accumulation associated with bleomycin-induced lung fibrosis, only transplantation of transduced HSCs greatly attenuated the histological damage. Using double immunohistochemistry, we show that the reduced lung damage likely occurs through endogenous type II pneumocyte proliferation induced by KGF. Taken together, our data indicates that bone marrow transplantation of lentivirus-transduced HSCs can attenuate lung damage, and shows for the first time the potential of using an inducible Tet-On system for cell based gene therapy in the lung.

Fbw7 controls neural stem cell differentiation and progenitor apoptosis via Notch and c-Jun

Neural stem and progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes and oligodendrocytes. However, the mechanisms underlying the decision of a stem cell to either self-renew or differentiate are incompletely understood. We demonstrate here that Fbw7 (F-box and WD repeat domain containing-7), the substrate recognition component of an SCF (complex of SKP1, CUL1 and F-box protein)-type E3 ubiquitin ligase, is a key regulator of NSC/NPC viability and differentiation. The absence of Fbw7 in the mouse brain caused severely impaired stem cell differentiation and increased progenitor cell death. Fbw7 deficiency resulted in accumulation of two SCF(Fbw7) substrates, the transcription factors active Notch1 and N-terminally phosphorylated c-Jun. Genetic and pharmacological rescue experiments identified c-Jun as a key substrate of Fbw7 in controlling progenitor cell viability, whereas inhibition of Notch signaling alleviated the block in stem cell differentiation. Thus Fbw7 controls neurogenesis by antagonizing Notch and c-Jun N-terminal kinase (JNK)/c-Jun signaling.