Distinct Transcriptional Programs Research Articles

High pathogen burden in childhood promotes the development of unconventional innate-like CD8+ T cells.

Cellular and humoral constituents of the immune system differ significantly between children and adults, yet very little is known about the impact of early-life pathogen exposure on this immunologic transition. We examined CD4+ and CD8+ T cell subsets defined by CCR7 and CD45RA expression in two longitudinal pediatric cohorts experiencing divergent levels of pathogen burden. Using multiparameter flow cytometry, along with serological, cytokine, and transcriptomic data, we show that cumulative pathogen burden promotes the development of atypical CD8dim T cells with an innate-like profile (Granzyme Bhi, IFNγlow, TNFαlow, PLFZhi, ID2hi, IKZF2hi) in contrast to age-matched children residing in a low pathogen-exposure area who display a more conventional CD8bright profile (IFNγ+, TNFα+, CCL4+). Furthermore, these unconventional T cells had stunted proliferation, distinct transcriptional programs, and impaired T cell receptor signaling and were enriched in hallmark TNFα, NF-κB, and IL-6 gene signaling pathways, reminiscent of NK cells and type-1 innate lymphoid cells. Our findings suggest that these unconventional CD8dim T cells arise in a very particular immunological context and may provide a deeper understanding of the heterogeneity in human immune responses.

CREB Regulates Distinct Adaptive Transcriptional Programs in Astrocytes and Neurons

The cyclic AMP response element binding protein (CREB) is a primary hub of activity-driven genetic programs in neurons controlling plasticity, neurogenesis and survival. By contrast, the gene networks coordinated by CREB in astrocytes are unknown despite the fact that the astrocytic CREB is also activity-driven and neuroprotective. Herein we identified the transcriptional programs regulated by CREB in astrocytes as compared to neurons using, as study materials, transcriptome databases of astrocyte exposed to well-known activators of CREB-dependent transcription as well as publicly available transcriptomes of neuronal cultures. Functional CREB signatures were extracted from the transcriptomes using Gene Ontology, adult-brain gene lists generated by Translating Ribosome Affinity Purification (TRAP) and CREB-target gene repositories. We found minimal overlap between CREB signatures in astrocytes and neurons. In astrocytes, the top triad of functions regulated by CREB consists of ‘Gene expression’, ‘Mitochondria’, and ‘Signalling’, while in neurons it is ‘Neurotransmission’, ‘Signalling’ and ‘Gene expression’, the latter two being represented by different genes from those in astrocytes. The newly generated databases will provide a tool to explore novel means whereby CREB impinges on brain functions requiring adaptive, long-lasting changes by coordinating transcriptional cascades in astrocytes.

Epithelial-to-Pericyte Transition in Cancer.

During epithelial-to-mesenchymal transition (EMT), cells lose epithelial characteristics and acquire mesenchymal properties. These two processes are genetically separable and governed by distinct transcriptional programs, rendering the EMT outputs highly heterogeneous. Our recent study shows that the mesenchymal products generated by EMT often express multiple pericyte markers, associate with and stabilize blood vessels to fuel tumor growth, thus phenotypically and functionally resembling pericytes. Therefore, some EMT events represent epithelial-to-pericyte transition (EPT). The serum response factor (SRF) plays key roles in both EMT and differentiation of pericytes, and may inherently confer the pericyte attributes on EMT cancer cells. By impacting their intratumoral location and cell surface receptor expression, EPT may enable cancer cells to receive and respond to angiocrine factors produced by the vascular niche, and develop therapy resistance.

An imprinted non-coding genomic cluster at 14q32 defines clinically relevant molecular subtypes in osteosarcoma across multiple independent datasets

BackgroundA microRNA (miRNA) collection on the imprinted 14q32 MEG3 region has been associated with outcome in osteosarcoma. We assessed the clinical utility of this miRNA set and their association with methylation status.MethodsWe integrated coding and non-coding RNA data from three independent annotated clinical osteosarcoma cohorts (n = 65, n = 27, and n = 25) and miRNA and methylation data from one in vitro (19 cell lines) and one clinical (NCI Therapeutically Applicable Research to Generate Effective Treatments (TARGET) osteosarcoma dataset, n = 80) dataset. We used time-dependent receiver operating characteristic (tdROC) analysis to evaluate the clinical value of candidate miRNA profiles and machine learning approaches to compare the coding and non-coding transcriptional programs of high- and low-risk osteosarcoma tumors and high- versus low-aggressiveness cell lines. In the cell line and TARGET datasets, we also studied the methylation patterns of the MEG3 imprinting control region on 14q32 and their association with miRNA expression and tumor aggressiveness.ResultsIn the tdROC analysis, miRNA sets on 14q32 showed strong discriminatory power for recurrence and survival in the three clinical datasets. High- or low-risk tumor classification was robust to using different microRNA sets or classification methods. Machine learning approaches showed that genome-wide miRNA profiles and miRNA regulatory networks were quite different between the two outcome groups and mRNA profiles categorized the samples in a manner concordant with the miRNAs, suggesting potential molecular subtypes. Further, miRNA expression patterns were reproducible in comparing high-aggressiveness versus low-aggressiveness cell lines. Methylation patterns in the MEG3 differentially methylated region (DMR) also distinguished high-aggressiveness from low-aggressiveness cell lines and were associated with expression of several 14q32 miRNAs in both the cell lines and the large TARGET clinical dataset. Within the limits of available CpG array coverage, we observed a potential methylation-sensitive regulation of the non-coding RNA cluster by CTCF, a known enhancer-blocking factor.ConclusionsLoss of imprinting/methylation changes in the 14q32 non-coding region defines reproducible previously unrecognized osteosarcoma subtypes with distinct transcriptional programs and biologic and clinical behavior. Future studies will define the precise relationship between 14q32 imprinting, non-coding RNA expression, genomic enhancer binding, and tumor aggressiveness, with possible therapeutic implications for both early- and advanced-stage patients.

RLR-mediated immunity is unique and critical in the response to West Nile Virus

Abstract RIG-I-Like Receptors (RLRs) include RIG-I, MDA5, and LGP2. RLRs are vital pathogen recognition receptors in the defense against RNA viruses. West Nile Virus (WNV) is an emerging, neurotropic flavivirus that infects the central nervous system to cause disease and death. RIG-I and MDA5 work in tandem to initiate innate immune responses against WNV infection, though the role of LGP2 in this process is not well defined. To gain a deeper understanding of the contributions of each RLR in the innate immune response to WNV, we conducted a systems biology approach to assess the role of each RLR in immune programming. We conducted genome-wide RNAseq and bioinformatics analysis of WNV infection in bone marrow derived macrophages from the RLR-deficient mice. Here we show that the RLRs drive distinct immune gene activation and polarization of the immune response. In our data, the RLR-dependent, WNV-induced immune response polarization overshadows the classical drivers of viral innate immune responses, interferon I (IFN) and IFN-stimulated genes, thus underscoring the importance of innate immune activation for channeling the adaptive immune system into specific effector pathways. Bioinformatics analysis and computational modeling was used to define a unique gene signature of each RLR following WNV infection. Further, we dissected the complex virus-induced cytokine response utilizing multiplex analyses to link gene induction and cytokine protein output. These analyses reveal that distinct RLR-driven transcriptional programs lead to differential functional output in immune response initiation against WNV infection. Our study demonstrates a clear, defined role for RIG-I, MDA5, and LGP2 in immune programming against virus infection.

Gene-enhancer variants reveal diverse TCR-mediated differentiation.

The transcription factor IRF4 acts as a 'rheostat' for TCR signaling. Discrete levels of IRF4 can activate distinct transcriptional programs in T cells due to binding sites of variable affinity in groups of target genes.

Targeting epigenetic regulation of malaria blood‐stage infections

Epigenetic mechanisms control a variety of critical processes through the life‐cycle of the malaria parasite P. falciparum. In the blood‐stage of infection, this includes antigenic variation of proteins on the surface of parasite‐infected red blood cells that enable sequestration within the host microvasculature and organs, as well as the expression of genes required for transition to the transmissible sexual stage of the parasite. Key histone‐modifying enzymes have been identified as regulators of distinct transcriptional programs underlying these processes. We have focused our research on the histone deacetylases (HDACs) of P. falciparum, that enzymatically remove the acetyl group from histones to regulate transcription of specific gene sets. P. falciparum expresses one class I HDAC, two class II HDACs and two class III HDACs. We have been studying the functions of these HDACs in blood‐stage infections, to establish how they regulate different transcriptional programs and cellular processes in a ‘division of labor’. We are exploring how we can chemically target the essential functions of the P. falciparum HDACs to disrupt and halt proliferation and transmission of this deadly parasite.Support or Funding InformationBurroughs Wellcome Fund

The integrative metabolomic-transcriptomic landscape of glioblastome multiforme.

The purpose of this study was to map the landscape of metabolic-transcriptional alterations in glioblastoma multiforme. Omic-datasets were acquired by metabolic profiling (1D-NMR spectroscopy n=33 Patient) and transcriptomic profiling (n=48 Patients). Both datasets were analyzed by integrative network modeling. The computed model concluded in four different metabolic-transcriptomic signatures containing: oligodendrocytic differentiation, cell-cycle functions, immune response and hypoxia. These clusters were found being distinguished by individual metabolism and distinct transcriptional programs. The study highlighted the association between metabolism and hallmarks of oncogenic signaling such as cell-cycle alterations, immune escape mechanism and other cancer pathway alterations. In conclusion, this study showed the strong influence of metabolic alterations in the wide scope of oncogenic transcriptional alterations.

Epigenetic Basis of Neuronal and Synaptic Plasticity.

Neuronal network and plasticity change as a function of experience. Altered neural connectivity leads to distinct transcriptional programs of neuronal plasticity-related genes. The environmental challenges throughout life may promote long-lasting reprogramming of gene expression and the development of brain disorders. The modifications in neuronal epigenome mediate gene-environmental interactions and are required for activity-dependent regulation of neuronal differentiation, maturation and plasticity. Here, we highlight the latest advances in understanding the role of the main players of epigenetic machinery (DNA methylation and demethylation, histone modifications, chromatin-remodeling enzymes, transposons, and non-coding RNAs) in activity-dependent and long- term neural and synaptic plasticity. The review focuses on both the transcriptional and post-transcriptional regulation of gene expression levels, including the processes of promoter activation, alternative splicing, regulation of stability of gene transcripts by natural antisense RNAs, and alternative polyadenylation. Further, we discuss the epigenetic aspects of impaired neuronal plasticity and the pathogenesis of neurodevelopmental (Rett syndrome, Fragile X Syndrome, genomic imprinting disorders, schizophrenia, and others), stressrelated (mood disorders) and neurodegenerative Alzheimer's, Parkinson's and Huntington's disorders. The review also highlights the pharmacological compounds that modulate epigenetic programming of gene expression, the potential treatment strategies of discussed brain disorders, and the questions that should be addressed during the development of effective and safe approaches for the treatment of brain disorders.

Massively Parallel Single Cell RNA-Seq of Primary Lymphomas Reveals Distinct Cellular Lineages and Diverse, Intratumoral Transcriptional States

Follicular lymphoma (FL) is a generally incurable B-cell malignancy which has the potential to transform into highly aggressive lymphomas. Genomic studies indicate it is often a small subpopulation rather than the dominant population in the FL that gives rise to the more aggressive subtype.To resolve the underlying transcriptional networks of follicular B-cell lymphomas at single molecule and cell resolution, we leveraged droplet-based barcoding technology for highly parallel single cell RNA-Seq. We analyzed the transcriptomes from tens of thousands of cells derived from five primary FL tumors (average > 5,000 cells/sample). Simultaneously, we conducted multi-dimensional flow cell sorting to validate our characterizing of cellular lineages and critical expressed proteins. For each tumor, we identified multiple cellular subpopulations, matching known hematopoietic lineages. Despite some common features, such as MYC and BCLoverexpression, distinct transcriptional patterns and regulatory programs were evident among the different tumors. Within each tumor, subpopulations of malignant cell transcripts were compared to matched normal B-cells (Figure 1).Transcriptome analysis of >13,500 B-cells across 5 samples reveals sample-specific and joint clusters of B-cells. Each dot represents the t-SNE projection of a single cell’s transcriptome profile. Single cells are colored according to sample origin (control PBMC samples 1 & 2; follicular lymphoma samples 1, 2 & 3). Normal B-cells within follicular lymphoma samples cluster together with PBMC derived B-cells. Malignant B-cells cluster according to sample origin, except for the cycling tumor cells (upper left cell cluster).Malignant B-cells were characterized by expression of restricted immunoglobulin light chain type (either kappa or lambda), BCL2 expression, and CD20 expression. We show evidence for the coexistence of small, malignant subpopulations alongside the dominant FL population in the majority of tumors. These smaller subpopulations harbored several transcriptional changes that were absent from the dominant population, including upregulation of MHC class II expression, downregulation of β-2-microglobulin, and exclusive expression of chemokines (CCL22 and CCL17), that would have resulted in alteration in antigen presentation and of the T cell immune system. In addition, we characterize the transcriptomes of the infiltrating immune cell populations that are characteristic of this disease. Our findings provide an unprecedented resolution of distinct immune lineages as seen by transcriptionally characterized cellular diversity. [Display omitted] DisclosuresLevy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

The Use of Chromatin Precipitation Coupled to DNA Sequencing (ChIP-Seq) for the Analysis of Zta Binding to the Human and EBV Genome.

Determining which components of the transcription machinery associate with the viral and cellular genome, and how this changes at specific stages of the viral life cycle is paramount to understanding how the distinct transcriptional programs associated with primary infection, latency, and disease are established and how they are reprogrammed during initiation and execution of the viral lytic replication cycle. Chromatin precipitations linked to next generation DNA sequencing (ChIP-Seq) allow for the interactions of proteins with DNA to be mapped across both viral and cellular genomes. This can be applied to viral and cellular transcription factors, coactivators and corepressors, modified histones, and modulators of chromatin.

Palmitate-induced ER stress increases trastuzumab sensitivity in HER2/neu-positive breast cancer cells

BackgroundHER2/neu-positive breast cancer cells have recently been shown to use a unique Warburg-like metabolism for survival and aggressive behavior. These cells exhibit increased fatty acid synthesis and storage compared to normal breast cells or other tumor cells. Disruption of this synthetic process results in apoptosis. Since the addition of physiological doses of exogenous palmitate induces cell death in HER2/neu-positive breast cancer cells, the pathway is likely operating at its limits in these cells. We have studied the response of HER2/neu-positive breast cancer cells to physiological concentrations of exogenous palmitate to identify lipotoxicity-associated consequences of this physiology. Since epidemiological data show that a diet rich in saturated fatty acids is negatively associated with the development of HER2/neu-positive cancer, this cellular physiology may be relevant to the etiology and treatment of the disease. We sought to identify signaling pathways that are regulated by physiological concentrations of exogenous palmitate specifically in HER2/neu-positive breast cancer cells and gain insights into the molecular mechanism and its relevance to disease prevention and treatment.MethodsTranscriptional profiling was performed to assess programs that are regulated in HER2-normal MCF7 and HER2/neu-positive SKBR3 breast cancer cells in response to exogenous palmitate. Computational analyses were used to define and predict functional relationships and identify networks that are differentially regulated in the two cell lines. These predictions were tested using reporter assays, fluorescence-based high content microscopy, flow cytometry and immunoblotting. Physiological effects were confirmed in HER2/neu-positive BT474 and HCC1569 breast cancer cell lines.ResultsExogenous palmitate induces functionally distinct transcriptional programs in HER2/neu-positive breast cancer cells. In the lipogenic HER2/neu-positive SKBR3 cell line, palmitate induces a G2 phase cell cycle delay and CHOP-dependent apoptosis as well as a partial activation of the ER stress response network via XBP1 and ATF6. This response appears to be a general feature of HER2/neu-positive breast cancer cells but not cells that overexpress only HER2/neu. Exogenous palmitate reduces HER2 and HER3 protein levels without changes in phosphorylation and sensitizes HER2/neu-positive breast cancer cells to treatment with the HER2-targeted therapy trastuzumab.ConclusionsSeveral studies have shown that HER2, FASN and fatty acid synthesis are functionally linked. Exogenous palmitate exerts its toxic effects in part through inducing ER stress, reducing HER2 expression and thereby sensitizing cells to trastuzumab. These data provide further evidence that HER2 signaling and fatty acid metabolism are highly integrated processes that may be important for disease development and progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2611-8) contains supplementary material, which is available to authorized users.

Sarcoidosis activates diverse transcriptional programs in bronchoalveolar lavage cells.

BackgroundSarcoidosis is a multisystem immuno-inflammatory disorder of unknown etiology that most commonly involves the lungs. We hypothesized that an unbiased approach to identify pathways activated in bronchoalveolar lavage (BAL) cells can shed light on the pathogenesis of this complex disease.MethodsWe recruited 15 patients with various stages of sarcoidosis and 12 healthy controls. All subjects underwent bronchoscopy with lavage. For each subject, total RNA was extracted from BAL cells and hybridized to an Affymetrix U133A microarray. Rigorous statistical methods were applied to identify differential gene expression between subjects with sarcoidosis vs. controls. To better elucidate pathways differentially activated between these groups, we integrated network and gene set enrichment analyses of BAL cell transcriptional profiles.ResultsSarcoidosis patients were either non-smokers or former smokers, all had lung involvement and only two were on systemic prednisone. Healthy controls were all non-smokers. Comparison of BAL cell gene expression between sarcoidosis and healthy subjects revealed over 1500 differentially expressed genes. Several previously described immune mediators, such as interferon gamma, were upregulated in the sarcoidosis subjects. Using an integrative computational approach we constructed a modular network of over 80 gene sets that were highly enriched in patients with sarcoidosis. Many of these pathways mapped to inflammatory and immune-related processes including adaptive immunity, T-cell signaling, graft vs. host disease, interleukin 12, 23 and 17 signaling. Additionally, we uncovered a close association between the proteasome machinery and adaptive immunity, highlighting a potentially important and targetable relationship in the pathobiology of sarcoidosis.ConclusionsBAL cells in sarcoidosis are characterized by enrichment of distinct transcriptional programs involved in immunity and proteasomal processes. Our findings add to the growing evidence implicating alveolar resident immune effector cells in the pathogenesis of sarcoidosis and identify specific pathways whose activation may modulate disease progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0411-y) contains supplementary material, which is available to authorized users.

Abstract 3692: Transcriptome analysis of oral tongue cancer reveals novel insights into wild type and mutant TP53 transcription program

Abstract The p53 oncoprotein is a tumor suppressor that is stabilized upon various forms of cellular stresses to induce transcription of genes regulating cell cycle arrest or apoptosis. TP53 is the most frequently mutated gene in human cancers and majority of mutations are located in the region encoding the DNA binding domain compromising thereby its transcription activation ability and resulting in loss of function. Recent studies however suggest mutant p53 proteins to exhibit a gain of function property. Specific p53 missense mutations can result in an altered transcription program causing positive regulation of cell proliferation, metastasis and chemoresistance. In our previous studies, we performed comprehensive characterization of squamous cell carcinoma of the oral tongue (SCCOT) with respect to p53, EGFR, Wnt, MSI, LoH of several tumor suppressor loci and HPV status. Mutant p53 was a significant predictor of overall survival and the TP53 codon 72 Proline allele was significantly associated with SCCOT. In order to dissect the role of mutant p53 in tongue cancer, we performed genome wide DNA and RNA profiling of 26 and 40 SCCOT samples, respectively. Both mutant and wild type tumor samples appeared to exhibit comparable levels of DNA copy number alterations. Transcriptome data analyses using a combination of single sample gene set enrichment analysis and comparative marker selection revealed gene sets that could significantly distinguish p53 mutant and wild type tumor samples. Significance analysis of microarrays performed on all genes constituting the differentially enriched gene sets surprisingly identified only two genes to be upregulated in p53 mutant samples at a false discovery rate significantly lower than 10% namely TP53 itself and SMARCD1; the latter a member of the SWI/SNF chromatin remodelling complex. Elevated levels of TP53 transcript in tumors harbouring mutant p53 significantly correlated with levels of ZMAT3, itself induced by p53 and known to stabilize the TP53 transcript. In addition, the analysis revealed several known (ATF3 and others) and novel (GCHFR and others) targets of wild type p53. Differential expression of all targets was validated in additional tongue cancer samples. Ectopic expression of certain (but not all) p53 mutant proteins in p53 null cells induced SMARCD1 (but not canonical wild type p53 targets) while expression of wild type p53 induced GCHFR, ATF3, CDKN1A, etc. (but not SMARCD1). In contrast, p53 stabilization in cells harboring wild type p53 caused elevation of GCHFR, ATF3, CDKN1A, etc., but not of SMARCD1. Validation of novel targets using promoter-luciferase constructs, chromatin immunoprecipitation PCR and a tongue cancer tissue microarray is underway. This is perhaps the first evidence from Head and Neck tumor samples for a gain of function activity of mutant p53. Thus wild type and mutant p53 may support distinct transcription programs in tongue cancer. Citation Format: Raju SR Adduri, Padmavathi Kavadipula, Viswakalyan Kotapalli, Leena Bashyam, Anupama Shirke, Arun kumar Paripati, Swarnalata Gowrishankar, Mukta Srinivasulu, Mohammed Mujtaba Ali, Subramanyeshwar Rao, Snehalatha Dhagam, Mohana Vamsy Chigurupati, Shantveer G. Uppin, Vijaya Tourani, Murali D. Bashyam. Transcriptome analysis of oral tongue cancer reveals novel insights into wild type and mutant TP53 transcription program. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3692.

Abstract LB-321: TP63 isoforms regulate distinct and cooperative transcriptional signatures that drive cancer progression and predict clinical outcomes

Abstract TP63 is required to maintain stem cell pluripotency and suppresses the metastatic potential of cancer cells through multiple mechanisms. Even though roles for Tp63 in cancer have been described, the transcriptional roles of TP63 isoforms, namely TAp63 and ΔNp63, in tumor development and progression have been perplexing due to their variable expression in tissues, their complex interaction with each other and with TP53, and the lack of adequate antibodies to distinguish between their isoforms. Of concern is the use of total p63 as a diagnostic marker for many epithelial cancers in the clinic with disregard of the existence and functions of the various isoforms, which may lead to improper diagnosis. These notions necessitate a deeper understanding of how the distinct transcriptional programs controlled by TP63 isoforms affect cancer progression and patient outcomes. To shed light on the activities of the TP63 isoforms in cancer development and progression, we conducted a pan-cancer analysis of The Cancer Genome Atlas (TCGA) to identify the transcriptional networks regulated by TAp63 and ΔNp63 using transcriptomes derived from epidermal cells of TAp63-/- and ΔNp63-/- mice. We then derived 17 cancer developmental (tumor versus corresponding normal) and 27 cancer progression (high stage versus low stage) signatures. Our analysis revealed a consistent tumor suppressive pattern for TAp63. In contrast, we identified pleiotropic roles for ΔNp63 in tumor development and progression. Our findings revealed a cooperative role between TAp63 and ΔNp63 via their gene signatures that predict survival and correlates with progression of cancer patients in bladder cancer (BLCA), clear cell renal cell carcinoma (KIRC), papillary renal cell carcinoma (KIRP) and low grade glioma. Importantly, we found that ΔNp63 transcriptional activity integrates the remodeling of extracellular matrix remodeling to provide an ideal environment in these cancers to undergo epithelial to mesenchymal transition and gain pluripotent stem cell characteristics, thus supporting tumor progression, while TAp63 activity acts to regulate the cell cycle required in stem cell maintenance which may further allow tumors to progress. These signatures are not only predictive of survival across stages, but can even stratify patients within the same stage into different survival groups in the genitourinary tumors as well as in low grade gliomas. We then validated our findings in independent cohorts. Our data describe a global approach for understanding transcriptional activities of TP63 isoforms across a large number of cancer types, potentially enabling the identification of patient subsets most likely to benefit from therapies predicated on manipulating specific TP63 isoforms in order to inhibit the acquisition of pluripotency potential. Citation Format: Hussein Abbas, Kimal Rajapakshe, Justin Wong, Ngoc Hoang Bao Bui, Preethi Gunaratne, Kenneth Tsai, Cristian Coarfa, Elsa Flores. TP63 isoforms regulate distinct and cooperative transcriptional signatures that drive cancer progression and predict clinical outcomes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-321.

Endothelial HO-1 induction by model TG-rich lipoproteins is regulated through a NOX4-Nrf2 pathway

Circulating levels of chylomicron remnants (CMRs) increase postprandially and their composition directly reflects dietary lipid intake. These TG-rich lipoproteins likely contribute to the development of endothelial dysfunction, albeit via unknown mechanisms. Here, we investigated how the FA composition of CMRs influences their actions on human aortic endothelial cells (HAECs) by comparing the effects of model CMRs—artificial TG-rich CMR-like particles (A-CRLPs)—containing TGs extracted from fish, DHA-rich algal, corn, or palm oils. HAECs responded with distinct transcriptional programs according to A-CRLP TG content and oxidation status, with genes involved in antioxidant defense and cytoprotection most prominently affected by n-3 PUFA-containing A-CRLPs. These particles were significantly more efficacious inducers of heme oxygenase-1 (HO-1) than n-6 PUFA corn or saturated FA-rich palm CRLPs. Mechanistically, HO-1 induction by all CRLPs requires NADPH oxidase 4, with PUFA-containing particles additionally dependent upon mitochondrial reactive oxygen species. Activation of both p38 MAPK and PPARβ/δ culminates in increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression/nuclear translocation and HO-1 induction. These studies define new molecular pathways coupling endothelial cell activation by model CMRs with adaptive regulation of Nrf2-dependent HO-1 expression and may represent key mechanisms through which dietary FAs differentially impact progression of endothelial dysfunction.

Distinct Transcriptional Programs Control Cross-Priming in Classical and Monocyte-Derived Dendritic Cells.

Both classical DCs (cDCs) and monocyte-derived DCs (Mo-DCs) are capable of cross-priming CD8(+) Tcells in response to cell-associated antigens. Wefound that Ly-6C(hi)TREML4(-) monocytes can differentiate into Zbtb46(+) Mo-DCs in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) but that Ly-6C(hi)TREML4(+) monocytes were committed to differentiate into Ly-6C(lo)TREML4(+) monocytes. Differentiation of Zbtb46(+) Mo-DCs capable of efficient cross-priming required both GM-CSF and IL-4 and was accompanied by the induction of Batf3 and Irf4. However, monocytes require IRF4, but not BATF3, to differentiate into Zbtb46(+) Mo-DCs capable of cross-priming CD8(+) Tcells. Instead, Irf4(-/-) monocytes differentiate into macrophages in response to GM-CSF and IL-4. Thus, cDCs and Mo-DCs require distinct transcriptional programs of differentiation in acquiring the capacity to prime CD8(+) Tcells. These differences may be of consideration in the use of therapeutic DC vaccines based on Mo-DCs.

Classical and monocyte-derived DCs require distinct transcriptional programs for cross-presentation.

Abstract Classical DCs (cDCs) and monocyte-derived DCs (Mo-DCs) cross-present cell associated antigens, but whether these cells use a universal program for this process is unknown. In examining the requirements for cross-presentation by Mo-DCs, we discovered that TremL4 identifies two Ly-6Chimonocyte populations, of which only Ly-6ChiTremL4− monocytes can differentiate into Zbtb46+ Mo-DCs in response to GM-CSF and IL-4 in vitro. By contrast, Ly-6Chi TremL4+ monocytes lost Mo-DC potential and instead were committed to development of Ly-6Clo TremL4+ monocytes. Further, we found that differentiation of monocytes with GM-CSF required addition of IL-4 to generate Zbtb46+ Mo-DCs that cross-presented as efficiently as CD8α+ cDCs in vitro. However, unlike CD8α+ cDCs, Mo-DCs required only IRF4, and not Batf3, to cross-present cell-associated antigens. Further, Irf4−/− monocytes treated with GM-CSF and IL-4 could not differentiate into Zbtb46+ Mo-DCs, and instead developed into macrophages. Thus, distinct transcriptional programs are used by CD8α+ cDCs and Mo-DCs for cross-presentation which may drive different antigen-processing pathways.

The pruritus- and TH2-associated cytokine IL-31 promotes growth of sensory nerves

Pruritus is a cardinal symptom of atopic dermatitis, and an increased cutaneous sensory network is thought to contribute to pruritus. Although the immune cell-IL-31-neuron axis has been implicated in severe pruritus during atopic skin inflammation, IL-31's neuropoietic potential remains elusive. We sought to analyze the IL-31-related transcriptome in sensory neurons and to investigate whether IL-31 promotes sensory nerve fiber outgrowth. Invitro primary sensory neuron culture systems were subjected to whole-transcriptome sequencing, ingenuity pathway analysis, immunofluorescence, and nerve elongation, as well as branching assays after IL-31 stimulation. Invivo we investigated the cutaneous sensory neuronal network in wild-type, Il31-transgenic, and IL-31 pump-equipped mice. Transgenic Il31 overexpression and subcutaneously delivered IL-31 induced an increase in the cutaneous nerve fiber density in lesional skin invivo. Transcriptional profiling of IL-31-activated dorsal root ganglia neurons revealed enrichment for genes promoting nervous system development and neuronal outgrowth and negatively regulating cell death. Moreover, the growth cones of primary small-diameter dorsal root ganglia neurons showed abundant IL-31 receptor α expression. Indeed, IL-31 selectively promoted nerve fiber extension only in small-diameter neurons. Signal transducer and activator of transcription 3 phosphorylation mediated IL-31-induced neuronal outgrowth, and pharmacologic inhibition of signal transducer and activator of transcription 3 completely abolished this effect. In contrast, transient receptor potential cation channel vanilloid subtype 1 channels were dispensable for IL-31-induced neuronal sprouting. The pruritus- and TH2-associated novel cytokine IL-31 induces a distinct transcriptional program in sensory neurons, leading to nerve elongation and branching both invitro and invivo. This finding might help us understand the clinical observation that patients with atopic dermatitis experience increased sensitivity to minimal stimuli inducing sustained itch.

Mitochondria operate as signaling platforms in yeast aging

Mitochondria are vital to physiology and health of eukaryotic organisms across phyla. These organelles generate the majority of cellular ATP and create biosynthetic intermediates for amino acids, nucleotides and lipids [1]. Mitochondria can also operate as signaling platforms and structural scaffolds for coordinating diverse cellular responses to changes in a variety of physiological conditions [2, 3]. Therefore, the functional state of mitochondria is crucial for a plethora of cellular processes, including cell growth, division, differentiation, homeostasis, metabolism, stress response, signaling, immune response, survival and death [1 - 4]. Because mitochondrial functionality gradually declines with age in evolutionarily distant organisms, such age-related deterioration of mitochondrial function is regarded as the universal hallmark of cellular and organismal aging [5]. The budding yeast Saccharomyces cerevisiae, a unicellular eukaryote, has been intensively used as a model organism for uncovering mechanisms linking mitochondrial functionality and cellular aging [3]. Our recent studies have revealed how a geroprotective chemical compound delays yeast chronological aging by causing an age-related remodeling of mitochondrial lipidome [6], how such remodeling elicits changes to mitochondrial morphology and functionality [6], and how such changes enable mitochondria to operate as signaling platforms that exploit a distinct set of transcription factors to choreograph a longevity-extending transcriptional program for many nuclear genes [7]. We found that exogenously added lithocholic bile acid (LCA), which is known to slow the chronological aging of S. cerevisiae, enters yeast cell, amasses within a double membrane delimiting mitochondria and resides primarily in the inner mitochondrial membrane [6]. After being confined to mitochondrial membranes, LCA elicits specific changes in the concentrations of mitochondrial membrane phospholipids; these changes occur in an age-related fashion and are believed to be due to a characteristic remodeling of pathways for phospholipid synthesis and movement within both mitochondrial membranes [6]. The resulting major changes in mitochondrial membrane lipidome cause a substantial enlargement of mitochondria, decrease the number of these organelles, significantly increase the total length on mitochondrial cristae, and reduce the extent of connectivity between these cristae and the inner mitochondrial membrane [6]. These extensive alterations in mitochondrial abundance and morphology incite a distinct pattern of changes in the age-related chronology of such key longevity-defining processes as mitochondrial respiration, membrane potential maintenance, ATP synthesis and reactive oxygen species homeostasis [6]. Such specific changes to the age-related chronology of mitochondrial functionality allow mitochondria to function as signaling platforms that coordinate a stepwise establishment of a distinct transcriptional program for many nuclear genes [7]. The observed age-related rewiring of transcriptional patterns is choreographed by a distinct set of transcription factors [7]. These transcription factors respond to different aspects of altered mitochondrial functionality in yeast cells exposed to LCA; they include the following regulators of transcription: 1) the Rtg1, Rtg2 and Rtg3 protein components of the mitochondrial retrograde signaling pathway; 2) the Sfp1 protein component of the mitochondrial back-signaling pathway; 3) the Aft1 transcription factor modulated by the concentrations of iron-sulfur clusters, which function as inorganic cofactors of numerous mitochondrial, nuclear and cytosolic proteins; 4) a transcription factor involved in the unfolded protein response pathway of mitochondria-to-nucleus communications; this pathway is elicited in response to reduced concentrations of the mitochondrial protease Yme1 and the mitochondrial peptide exporter Mdl1; and 5) the transcription factors Yap1, Msn2/Msn4, Skn7 and Hog1, all modulated by mitochondrially generated reactive oxygen species [7]. Based on these observations, we proposed a model for how LCA-driven changes in mitochondrial proteome and functionality early and late in life of chronologically aging yeast coordinate a stepwise development of an aging-delaying cellular pattern and its maintenance throughout lifespan [7]. In sum, we demonstrated that the geroprotective chemical compound LCA can delay the onset and slow the progression of chronological aging in yeast by causing an age-related remodeling of mitochondrial lipidome. Our findings revealed that such remodeling of mitochondrial lipidome alters mitochondrial morphology and functionality, thereby enabling mito-chondria to act as signaling platforms that can 1) choreograph a longevity-extending transcriptional program for many nuclear genes governed by a distinct set of transcription factors; and 2) orchestrate a gradual establishment and long-term maintenance of a longevity-extending cellular pattern. Thus, targeting the discovered role of mitochondria as signaling platforms in yeast aging has potential as a novel therapeutic strategy for slowing aging, improving health, attenuating age-related pathologies and delaying the onset of age-related diseases in humans.