Microbial ligand-independent regulation of lymphopoiesis by NOD1.

Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. Here we demonstrate that NOD1, a cytosolic innate sensor of bacterial peptidoglycan, also functions in murine hematopoietic cells as a major regulator of both the generation and differentiation of lymphoid progenitors as well as peripheral T lymphocyte homeostasis. We further show that NOD1 mediates these functions by facilitating STAT5 signaling downstream of hematopoietic cytokines. In steady-state, loss of NOD1 resulted in a modest but significant decrease in numbers of mature T, B and natural killer cells. During systemic protozoan infection this defect was markedly enhanced, leading to host mortality. Lack of functional NOD1 also impaired T cell-dependent anti-tumor immunity while preventing colitis. These findings reveal that, in addition to its classical role as a bacterial ligand receptor, NOD1 plays an important function in regulating adaptive immunity through interaction with a major host cytokine signaling pathway.

Antisense transcription from lentiviral gene targeting linked to an integrated stress response in colorectal cancer cells.

Advances in gene therapy research have resulted in the successful development of new therapies for clinical use. Here, we explored a gene targeting approach to deplete ephrinB2 from colorectal cancer cells using an inducible lentiviral vector. EphrinB2, a transmembrane ephrin ligand, promotes colorectal cancer cell growth and viability and predicts poor patient survival when expressed at high levels in colorectal cancer tissues. We discovered that lentiviral vector integration and expression in the host DNA frequently drive divergent host gene transcription, generating antisense reads coupled with splicing events and generation of chimeric vector/host transcripts. Antisense transcription of host DNA was linked to development of an integrated stress response and cell death. Despite recent successes, off-target effects remain a concern in genetic medicine. Our results provide evidence that divergent gene transcription is a previously unrecognized off-target effect of lentiviral vector integration with built-in properties for regulation of gene expression.

Inactivation of axon guidance molecule netrin-1 in human colorectal cancer by an epigenetic mechanism.

Netrin-1, the protein product of the NTN1 gene, is an axon guidance molecule implicated in regulation of cell survival and tumorigenesis. Expression of the netrin-1 receptors deleted in colorectal cancer (DCC) and uncoordinated 5 homolog (UNC5H) is frequently silenced in colorectal cancer (CRC) by either loss of heterozygosity or epigenetic mechanisms. However, netrin-1 expression and regulation in CRC are mostly unknown. Here, we report that NTN1 expression is significantly reduced in most CRC tissues compared to the adjacent normal intestinal mucosa, and that NTN1 DNA methylation is significantly higher in CRCs (24.6%) than in the adjacent normal intestinal mucosa (4.0%). In 6 CRC cell lines, NTN1 expression is low. Treatment with 5-Aza-2'-deoxycytidine increased expression of NTN1 in CRC cell lines, indicating that DNA methylation represses NTN1 transcription in CRCs. NTN1 DNA hypermethylation was significantly associated with advanced CRC disease. Median netrin-1 serum levels were significantly decreased in CRC patients (330.1 pg/mL) compared with normal individuals (438.6 pg/mL). Our results suggest that netrin-1 is a candidate biomarker for CRC.

Reusable Single Cell for Iterative Epigenomic Analyses.

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.

Bone marrow niches in myelodysplastic syndromes.

Genetic and epigenetic lesions within hematopoietic cell populations drive diverse hematological malignancies. Myelodysplastic syndromes (MDS) are a group of myeloid neoplasms affecting the hematopoietic stem cells characterized by recurrent genetic abnormalities, myelodysplasia (a pathological definition of abnormal bone marrow structure), ineffective hematopoiesis resulting in blood cytopenia, and a propensity to evolve into acute myelogenous leukemia. Although there is evidence that the accumulation of a set of genetic mutations is an essential event in MDS, there is an increased appreciation of the contribution of specific microenvironments, niches, in the pathogenesis of MDS and response to treatment. In physiologic hematopoiesis, niches are critical functional units that maintain hematopoietic stem and progenitor cells and regulate their maturation into mature blood cells. In MDS and other hematological malignancies, altered bone marrow niches can promote the survival and expansion of mutant hematopoietic clones and provide a shield from therapy. In this review, we focus on our understanding of the composition and function of hematopoietic niches and their role in the evolution of myeloid malignancies, with an emphasis on MDS.

Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth.

The tyrosine phosphatase SHP2 is oncogenic in cancers driven by receptor-tyrosine-kinases, and SHP2 inhibition reduces tumor growth. Here, we report that SHP2 is an essential promoter of endothelial cell survival and growth in the remodeling tumor vasculature. Using genetic and chemical approaches to inhibit SHP2 activity in endothelial cells, we show that SHP2 inhibits pro-apoptotic STAT3 and stimulates proliferative ERK1/2 signaling. Systemic SHP2 inhibition in mice bearing tumor types selected for SHP2-independent tumor cell growth promotes degeneration of the tumor vasculature and blood extravasation; reduces tumor vascularity and blood perfusion; and increases tumor necrosis. Reduction of tumor growth ensues, independent of SHP2 targeting in the tumor cells, blocking immune checkpoints, or recruiting macrophages. We also show that inhibiting the Angiopoietin/TIE2/AKT cascade magnifies the vascular and anti-tumor effects of SHP2 inhibition by blocking tumor endothelial AKT signaling, not a target of SHP2. Since the SHP2 and Ang2/TIE2 pathways are active in vascular endothelial cells of human melanoma and colon carcinoma, SHP2 inhibitors alone or with Ang2/TIE2 inhibitors hold promise to effectively target the tumor endothelium.

Iterative epigenomic analyses in the same single cell.

Gene expression in individual cells is epigenetically regulated by DNA modifications, histone modifications, transcription factors, and other DNA-binding proteins. It has been shown that multiple histone modifications can predict gene expression and reflect future responses of bulk cells to extracellular cues. However, the predictive ability of epigenomic analysis is still limited for mechanistic research at a single cell level. To overcome this limitation, it would be useful to acquire reliable signals from multiple epigenetic marks in the same single cell. Here, we propose a new approach and a new method for analysis of several components of the epigenome in the same single cell. The new method allows reanalysis of the same single cell. We found that reanalysis of the same single cell is feasible, provides confirmation of the epigenetic signals, and allows application of statistical analysis to identify reproduced reads using data sets generated only from the single cell. Reanalysis of the same single cell is also useful to acquire multiple epigenetic marks from the same single cells. The method can acquire at least five epigenetic marks: H3K27ac, H3K27me3, mediator complex subunit 1, a DNA modification, and a DNA-interacting protein. We can predict active signaling pathways in K562 single cells using the epigenetic data and confirm that the predicted results strongly correlate with actual active signaling pathways identified by RNA-seq results. These results suggest that the new method provides mechanistic insights for cellular phenotypes through multilayered epigenome analysis in the same single cells.

Vasculopathy and Coagulopathy Associated with SARS-CoV-2 Infection.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has resulted in > 500,000 deaths worldwide, including > 125,000 deaths in the U.S. since its emergence in late December 2019 and June 2020. Neither curative anti-viral drugs nor a protective vaccine is currently available for the treatment and prevention of COVID-19. Recently, new clinical syndromes associated with coagulopathy and vasculopathy have emerged as a cause of sudden death and other serious clinical manifestations in younger patients infected with SARS-CoV-2 infection. Angiotensin converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 and other coronaviruses, is a transmembrane protein expressed by lung alveolar epithelial cells, enterocytes, and vascular endothelial cells, whose physiologic role is to induce the maturation of angiotensin I to generate angiotensin 1-7, a peptide hormone that controls vasoconstriction and blood pressure. In this review, we provide the general context of the molecular and cellular mechanisms of SARS-CoV-2 infection with a focus on endothelial cells, describe the vasculopathy and coagulopathy syndromes in patients with SARS-CoV-2, and outline current understanding of the underlying mechanistic aspects.

Evidence for a Mesothelial Origin of Body Cavity Effusion Lymphomas.

Background: Primary effusion lymphoma (PEL) is a Kaposi's sarcoma herpes virus (KSHV)-induced lymphoma that typically arises in body cavities of HIV-infected patients. PEL cells are often co-infected with Epstein-Barr virus (EBV). "PEL-like" lymphoma is a KSHV-unrelated lymphoma that arises in body cavities of HIV-negative patients. "PEL-like" lymphoma is sometimes EBV positive. The derivation of PEL/"PEL-like" cells is unclear. Methods: Mesothelial cells were cultured from body cavity effusions of 23 patients. Cell proliferation, cytokine secretion, marker phenotypes, KSHV/EBV infection, and clonality were evaluated by standard methods. Gene expression was measured by quantitative polymerase chain reaction and immunoblotting. A mouse model of PEL (3 mice/group) was used to evaluate tumorigenicity. Results: We found that the mesothelia derived from six effusions of HIV-infected patients with PEL or other KSHV-associated diseases contained rare KSHV + or EBV + mesothelial cells. After extended culture (16-17 weeks), some mesothelial cells underwent a trans-differentiation process, generating lymphoid-type CD45 + /B220 + , CD5 + , CD27 + , CD43 + , CD11c + , and CD3 - cells resembling "B1-cells," most commonly found in mouse body cavities. These "B1-like" cells were short lived. However, long-term KSHV + EBV - and EBV + KSHV - clonal cell lines emerged from mesothelial cultures from two patients that were clonally distinct from the monoclonal or polyclonal B-cell populations found in the patients' original effusions. Conclusions: Mesothelial-to-lymphoid transformation is a newly identified in vitro process that generates "B1-like" cells and is associated with the emergence of long-lived KSHV or EBV-infected cell lines in KSHV-infected patients. These results identify mesothelial cultures as a source of PEL cells and lymphoid cells in humans.

Characterization of Semaphorin 6A-Mediated Effects on Angiogenesis Through Regulation of VEGF Signaling.

Angiogenesis identifies the process of endothelial cell sprouting and remodeling leading to the formation of new and functional blood vessels. Vascular expansion during development and in the adult mammal provides nutrients and oxygen to areas with increased need. Although many molecules and pathways have been identified as regulators of angiogenesis, aspects of this complex process remain unclear. Particularly undefined are the signals that orchestrate vessel survival and pruning once new blood vessels have sprouted. These poorly characterized aspects of angiogenesis need exploration. This chapter describes the experiments and methods enabling the characterization of Semaphorin 6A as a critical regulator of endothelial cell survival and vessel function.

EphrinB2 regulates the emergence of a hemogenic endothelium from the aorta.

Adult-type intraembryonic hematopoiesis arises from specialized endothelial cells of the dorsal aorta (DA). Despite the critical importance of this specialized endothelium for establishment of hematopoietic stem cells and adult hematopoietic lineages, the mechanisms regulating its emergence are incompletely understood. We show that EphrinB2, a principal regulator of endothelial cell function, controls the development of endothelium producing adult-type hematopoiesis. The absence of EphrinB2 impairs DA-derived hematopoiesis. Transmembrane EphrinB2 and its EphB4 receptor interact in the emerging DA, which transiently harbors EphrinB2(+) and EphB4(+) endothelial cells, thereby providing an opportunity for bi-directional cell-to-cell signaling to control the emergence of the hemogenic endothelium. Embryonic Stem (ES) cell-derived EphrinB2(+) cells are enriched with hemogenic endothelial precursors. EphrinB2 silencing impairs ES generation of hematopoietic cells but not generation of endothelial cells. The identification of EphrinB2 as an essential regulator of adult hematopoiesis provides important insight in the regulation of early hematopoietic commitment.

Identification of IL-23p19 as an endothelial proinflammatory peptide that promotes gp130-STAT3 signaling.

Interleukin-23 (IL-23), a heterodimeric cytokine composed of the unique p19 peptide (IL-23p19) and a peptide called IL-12p40, which is shared with IL-12, is implicated in Crohn's disease, rheumatoid arthritis, psoriasis, and other immune-mediated inflammatory diseases. Endothelial cells produce the IL-23p19 peptide in the absence of the IL-12p40 chain and thus do not make heterodimeric IL-23. We found that intercellular IL-23p19 increased the cell surface abundances of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells, which enhanced the attachment of leukocytes and increased their transendothelial migration. Intracellular p19 associated with the cytokine receptor subunit gp130 and stimulated the gp130-dependent activation of signal transducer and activator of transcription 3 (STAT3) signaling. Proinflammatory factors promoted the generation of IL-23p19 in endothelial cells. The adventitial capillaries of inflamed temporal arteries in patients with giant-cell arteritis (GCA) had endothelial p19 protein associated with gp130, but did not contain the IL-12p40 chain. Because adventitial capillaries are essential for the entry of inflammatory cells into arterial walls, these data suggest that p19 may contribute to GCA disease and could represent a therapeutic target. Our results provide evidence that IL-23p19 is a previously unrecognized endothelial proinflammatory peptide that promotes leukocyte transendothelial migration, advancing our current understanding of the complexities of inflammatory responses.

EphrinB2 controls vessel pruning through STAT1-JNK3 signalling.

Angiogenesis produces primitive vascular networks that need pruning to yield hierarchically organized and functional vessels. Despite the critical importance of vessel pruning to vessel patterning and function, the mechanisms regulating this process are not clear. Here we show that EphrinB2, a well-known player in angiogenesis, is an essential regulator of endothelial cell death and vessel pruning. This regulation depends upon phosphotyrosine-EphrinB2 signalling repressing c-jun N-terminal kinase 3 activity via STAT1. JNK3 activation causes endothelial cell death. In the absence of JNK3, hyaloid vessel physiological pruning is impaired, associated with abnormal persistence of hyaloid vessels, defective retinal vasculature and microphthalmia. This syndrome closely resembles human persistent hyperplastic primary vitreus (PHPV), attributed to failed involution of hyaloid vessels. Our results provide evidence that EphrinB2/STAT1/JNK3 signalling is essential for vessel pruning, and that defects in this pathway may contribute to PHPV.

Notch and TGFß: Functional partners facilitating tumor progression.

Cell signals integral to the tumor microenvironment influence cancer progression. Tumor-associated myeloid cells secrete pro-tumorigenic agents including, but not limited to, the potent cytokine transforming growth factor ß (TGFß). We have discovered a network of extrinsic signals including delta-like 4 (Dll4), Notch and TGFß, linking malignant cells and tumor-infiltrating myeloid cells, a nexus portending a clinically-relevant anticancer treatment.

Tumor-infiltrating myeloid cells activate Dll4/Notch/TGF-ß signaling to drive malignant progression.

Myeloid cells that orchestrate malignant progression in the tumor microenvironment offer targets for a generalized strategy to attack solid tumors. Through an analysis of tumor microenvironments, we explored an experimental model of lung cancer that uncovered a network of Dll4/Notch/TGF-ß1 signals that links myeloid cells to cancer progression. Myeloid cells attracted to the tumor microenvironment by the tumor-derived cytokines CCL2 and M-CSF expressed increased levels of the Notch ligand Dll4, thereby activating Notch signaling in the tumor cells and amplifying tumor-intrinsic Notch activation. Heightened Dll4/Notch signaling in tumor cells magnified TGF-ß-induced pSMAD2/3 signaling and was required to sustain TGF-ß-induced tumor cell growth. Conversely, Notch blockade reduced TGF-ß signaling and limited lung carcinoma tumor progression. Corroborating these findings, by interrogating RNAseq results from tumor and adjacent normal tissue in clinical specimens of human head and neck squamous carcinoma, we found evidence that TGF-ß/Notch crosstalk contributed to progression. In summary, the myeloid cell-carcinoma signaling network we describe uncovers novel mechanistic links between the tumor microenvironment and tumor growth, highlighting new opportunities to target tumors where this network is active.

Semaphorin 6A regulates angiogenesis by modulating VEGF signaling.

Formation of new vessels during development and in the mature mammal generally proceeds through angiogenesis. Although a variety of molecules and signaling pathways are known to underlie endothelial cell sprouting and remodeling during angiogenesis, many aspects of this complex process remain unexplained. Here we show that the transmembrane semaphorin6A (Sema6A) is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating the expression and signaling of VEGFR2, which is known to maintain endothelial cell viability by autocrine VEGFR signaling. The silencing of Sema6A in primary endothelial cells promotes cell death that is not rescued by exogenous VEGF-A or FGF2, attributable to the loss of prosurvival signaling from endogenous VEGF. Analyses of mouse tissues demonstrate that Sema6A is expressed in angiogenic and remodeling vessels. Mice with null mutations of Sema6A exhibit significant defects in hyaloid vessels complexity associated with increased endothelial cell death, and in retinal vessels development that is abnormally reduced. Adult Sema6A-null mice exhibit reduced tumor, matrigel, and choroidal angiogenesis compared with controls. Sema6A plays important roles in development of the nervous system. Here we show that it also regulates vascular development and adult angiogenesis.

BAZF, a novel component of cullin3-based E3 ligase complex, mediates VEGFR and Notch cross-signaling in angiogenesis.

Angiogenic homeostasis is maintained by a balance between vascular endothelial growth factor (VEGF) and Notch signaling in endothelial cells (ECs). We screened for molecules that might mediate the coupling of VEGF signal transduction with down-regulation of Notch signaling, and identified B-cell chronic lymphocytic leukemia/lymphoma6-associated zinc finger protein (BAZF). BAZF was induced by VEGF-A in ECs to bind to the Notch signaling factor C-promoter binding factor 1 (CBF1), and to promote the degradation of CBF1 through polyubiquitination in a CBF1-cullin3 (CUL3) E3 ligase complex. BAZF disruption in vivo decreased endothelial tip cell number and filopodia protrusion, and markedly abrogated vascular plexus formation in the mouse retina, overlapping the retinal phenotype seen in response to Notch activation. Further, impaired angiogenesis and capillary remodeling were observed in skin-wounded BAZF(-/-) mice. We therefore propose that BAZF supports angiogenic sprouting via BAZF-CUL3-based polyubiquitination-dependent degradation of CBF1 to down-regulate Notch signaling.

Oral ingestion of cow's milk immunoglobulin G stimulates some cellular immune systems and suppresses humoral immune responses in mouse.

Four-week-old male C3H/HeN mice were bred with diets consisting of ovalbumin alone (OVA, control diet) or mixtures of OVA and cow's milk immunoglobulin G (IgG-added diets) as a protein source for 4 or 5 weeks, and both the cellular and humoral immune properties of the mice were investigated. The number of interleukin (IL)-12+CD11b+ cells in spleens and the formation of superoxide by peritoneal macrophages were higher in mice given the IgG-added diet than in those given the control diet. The number of natural killer cells in Peyer's patches or spleens and the cytotoxic activity of spleen cells toward an erythroleukemia cell line, K562, were also higher in mice given the IgG-added diet. In contrast, the numbers of interferon-gamma+CD4+ and IL-4+CD4+ cells in Peyer's patches or spleens and the levels of total or OVA-specific intestinal IgA and serum IgG were significantly lower in mice given the IgG-added diet than in those given the control diet. In addition, the number of cells expressing CD19 in spleens was significantly higher in mice given the IgG-added diet. These results indicate that oral ingestion of cow's milk IgG may stimulate some innate cellular immune systems, while suppressing humoral adaptive immune responses in the mouse.