Adipocyte differentiation and function have become the major research targets due to the increasing interest in obesity and related metabolic conditions. Although, late stages of adipogenesis have been extensively studied, the early phases remain poorly understood. Here we present that supplementing ascorbic acid (AsA) to the adipogenic differentiation cocktail enables the robust and efficient differentiation of mouse embryonic stem cells (mESCs) to mature adipocytes. Such ESC-derived adipocytes mimic the gene-expression profile of subcutaneous isolated adipocytes in vivo remarkably well, much closer than 3T3-L1 derived ones. Moreover, the differentiated cells are in a monolayer, allowing a broad range of genome-wide studies in early and late stages of adipocyte differentiation to be performed.

Serotonin is a monoamine neurotransmitter that signals through a wide array of receptors (5-HT1-7) many of which are also involved in immune processes. Dendritic cells (DCs) are crucial players in immune defense by bridging innate and adaptive immune responses via their vast repertoire of pattern recognition receptors and antigen-presenting capability. Although serotonin is known to influence immunity at many levels, cell type-specific expression and function of its receptors remains poorly understood. Here we aimed to study 5-HT1-7 expression and function in CD1a- and CD1a+ human monocyte-derived DCs (moDCs). We found that the 5-HT2B receptor-subtype is solely expressed by the inflammatory CD1a+ moDC subset. Specific 5-HT2B activation potently inhibited TLR2, TLR3, and TLR7/8-induced proinflammatory cytokine and chemokine (TNF-α, IL-6, IL-8, IP-10, IL-12) but not type I interferon-β responses. 5-HT2B agonism also interfered with the polarization of CD1a+ moDC-primed CD4+ T cells towards inflammatory Th1 and Th17 effector lymphocytes. Here we report the subset-specific expression and immunomodulatory function of 5-HT2B in human moDCs. Our results expand the biological role of 5-HT2B which may act not only as a neurotransmitter receptor, but also as an important modulator of both innate and adaptive immune responses.

Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPARγ drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)-4. The functions of PPARγ on macrophage polarization in this setting were independent of ligand binding. Ligand-insensitive PPARγ bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL-4 re-stimulation. Ligand-insensitive PPARγ binding controlled the expression of an extracellular matrix remodeling-related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL-4 and PPARγ in the affected tissue. Thus, a predominantly ligand-insensitive PPARγ:RXR cistrome regulates progressive and/or reinforcing macrophage polarization.

Macrophages polarize into functionally distinct subtypes while responding to microenvironmental cues. The identity of proximal transcription factors (TFs) downstream from the polarization signals are known, but their activity is typically transient, failing to explain the long-term, stable epigenomic programs developed. Here, we mapped the early and late epigenomic changes of interleukin-4 (IL-4)-induced alternative macrophage polarization. We identified the TF, early growth response 2 (EGR2), bridging the early transient and late stable gene expression program of polarization. EGR2 is a direct target of IL-4-activated STAT6, having broad action indispensable for 77% of the induced gene signature of alternative polarization, including its autoregulation and a robust, downstream TF cascade involving PPARG. Mechanistically, EGR2 binding results in chromatin opening and the recruitment of chromatin remodelers and RNA polymerase II. Egr2 induction is evolutionarily conserved during alternative polarization of mouse and human macrophages. In the context of tissue resident macrophages, Egr2 expression is most prominent in the lung of a variety of species. Thus, EGR2 is an example of an essential and evolutionarily conserved broad acting factor, linking transient polarization signals to stable epigenomic and transcriptional changes in macrophages.

Phosphorus is one of the essential nutrients for plant growth, but it may be relatively unavailable to plants because of its chemistry. In soil, the majority of phosphorus is present in the form of a phosphate, usually as metal complexes making it bound to minerals or organic matter. Therefore, inorganic phosphate solubilization is an important process of plant growth promotion by plant associated bacteria and fungi. Non-nodulating plant species have been shown to thrive in low-nutrient environments, in some instances by relying on plant associated microorganisms called endophytes. These microorganisms live within the plant and help supply nutrients for the plant. Despite their potential enormous environmental importance, there are a limited number of studies looking at the direct molecular impact of phosphate solubilizing endophytic bacteria on the host plant. In this work, we studied the impact of two endophyte strains of wild poplar (Populus trichocarpa) that solubilize phosphate. Using a combination of x-ray imaging, spectroscopy methods, and proteomics, we report direct evidence of endophyte-promoted phosphorus uptake in poplar. We found that the solubilized phosphate may react and become insoluble once inside plant tissue, suggesting that endophytes may aid in the re-release of phosphate. Using synchrotron x-ray fluorescence spectromicroscopy, we visualized the nutrient phosphorus inside poplar roots inoculated by the selected endophytes and found the phosphorus in both forms of organic and inorganic phosphates inside the root. Tomography-based root imaging revealed a markedly different root biomass and root architecture for poplar samples inoculated with the phosphate solubilizing bacteria strains. Proteomics characterization on poplar roots coupled with protein network analysis revealed novel proteins and metabolic pathways with possible involvement in endophyte enriched phosphorus uptake. These findings suggest an important role of endophytes for phosphorus acquisition and provide a deeper understanding of the critical symbiotic associations between poplar and the endophytic bacteria.

Hematological and oncological disorders represent leading causes of childhood mortality. Neuropeptide somatostatin (SST) has been previously demonstrated in various pediatric tumors, but limited information exists on the expression and characteristics of SST receptors (SSTR) in hematological and oncological disorders of children. We aimed to investigate the expression of mRNA for SSTR subtypes (SSTR-1-5) in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of SSTRs were further studies by ligand competition assay. Our results show that the pediatric tumor samples highly expressed mRNA for the five SSTR subtypes with various patterns. The mRNA for SSTR-2 was detected in all specimens independently of their histological type. A Hodgkin lymphoma sample co-expressed mRNA for all five SSTR subtypes. SSTR-3 and SSTR-5 were detected only in malignant specimens, such as rhabdomyosarcoma, Hodgkin lymphoma, acute lymphoblastic leukemia, and a single nonmalignant condition, hereditary spherocytosis. The incidence of SSTR-1 and SSTR-4 was similar (60%) in the 15 specimens investigated. Radioligand binding studies demonstrated the presence of specific SSTRs and high affinity binding of SST analogs in pediatric solid tumors investigated. The high incidence of SSTRs in hematological and oncological disorders in children supports the merit of further investigation of SSTRs as molecular targets for diagnosis and therapy.

IL-4-driven alternative macrophage activation and proliferation are characteristic features of both antihelminthic immune responses and wound healing in contrast to classical macrophage activation, which primarily occurs during inflammatory responses. The signaling pathways defining the genome-wide microRNA expression profile as well as the cellular functions controlled by microRNAs during alternative macrophage activation are largely unknown. Hence, in the current work we examined the regulation and function of IL-4-regulated microRNAs in human and mouse alternative macrophage activation.

In addition to smoking, genetic predisposition is believed to play a major role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Genetic association studies of new candidate genes in COPD may lead to improved understanding of the pathogenesis of the disease.

Recently, we revealed the importance of follicular helper T cells (T(FH)) in the pathogenesis of primary Sjögren's syndrome (pSS). In the present study, we focused on the site of the inflammation and determined the composition of lymphocyte infiltration in labial salivary gland (LSG) biopsies with special emphasis on T(FH) and germinal center B cells. We selected tissue blocks obtained from ten patients at the time of disease onset. Detection of cell specific markers was performed with immunohistochemical and immunofluorescence stainings. We evaluated patients' clinical and laboratory features retrospectively and assessed the relation between disease course and early histopathological findings. LSG biopsies were graded based on the extension and arrangement level of periductal inflammatory cell infiltrates. T(FH) cell markers (CD84, PD-1, and Bcl-6) occurred predominantly in more organized structures with higher focus scores. The coexpression of CD3 and Bcl-6 markers clearly identified T(FH) cells close to Bcl-6(+) B cells with the typical formation of germinal centers. Systemic features were developed later in the disease course only in patients with highly structured infiltrates and the presence of T(FH) cells. Our observations suggest that the presence of T(FH) cells in LSGs at the disease onset may predict a more pronounced clinical course of pSS.

Dendritic cells (DCs) expressing CD1d, a molecule responsible for lipid antigen presentation, are capable of enhancing natural killer T (iNKT) cell proliferation. The signals controlling CD1 expression and lipid antigen presentation are poorly defined. We have shown previously that stimulation of the lipid-activated transcription factor, peroxisome proliferator-activated receptor (PPAR)gamma, indirectly regulates CD1d expression. Here we demonstrate that PPARgamma, turns on retinoic acid synthesis by inducing the expression of retinol and retinal metabolizing enzymes such as retinol dehydrogenase 10 and retinaldehyde dehydrogenase type 2 (RALDH2). PPARgamma-regulated expression of these enzymes leads to an increase in the intracellular generation of all-trans retinoic acid (ATRA) from retinol. ATRA regulates gene expression via the activation of the retinoic acid receptor (RAR)alpha in human DCs, and RARalpha acutely regulates CD1d expression. The retinoic acid-induced elevated expression of CD1d is coupled to enhanced iNKT cell activation. Furthermore, in vivo relevant lipids such as oxidized low-density lipoprotein can also elicit retinoid signaling leading to CD1d up-regulation. These data show that regulation of retinoid metabolism and signaling is part of the PPARgamma-controlled transcriptional events in DCs. The uncovered mechanisms allow the DCs to respond to altered lipid homeostasis by changing CD1 gene expression.

Tissue regeneration requires inflammatory and reparatory activity of macrophages. Macrophages detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of effector functions of macrophages coordinated with other cell types inside the injured tissue. The gene regulatory events supporting the sensory and effector functions of macrophages involved in tissue repair are not well understood. Here we show that the lipid activated transcription factor, PPARγ, is required for proper skeletal muscle regeneration, acting in repair macrophages. PPARγ controls the expression of the transforming growth factor-β (TGF-β) family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell fusion. This work establishes PPARγ as a required metabolic sensor and transcriptional regulator of repair macrophages. Moreover, this work also establishes GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair.

The concept of tissue-specific gene expression posits that lineage-determining transcription factors (LDTFs) determine the open chromatin profile of a cell via collaborative binding, providing molecular beacons to signal-dependent transcription factors (SDTFs). However, the guiding principles of LDTF binding, chromatin accessibility and enhancer activity have not yet been systematically evaluated. We sought to study these features of the macrophage genome by the combination of experimental (ChIP-seq, ATAC-seq and GRO-seq) and computational approaches. We show that Random Forest and Support Vector Regression machine learning methods can accurately predict chromatin accessibility using the binding patterns of the LDTF PU.1 and four other key TFs of macrophages (IRF8, JUNB, CEBPA and RUNX1). Any of these TFs alone were not sufficient to predict open chromatin, indicating that TF binding is widespread at closed or weakly opened chromatin regions. Analysis of the PU.1 cistrome revealed that two-thirds of PU.1 binding occurs at low accessible chromatin. We termed these sites labelled regulatory elements (LREs), which may represent a dormant state of a future enhancer and contribute to macrophage cellular plasticity. Collectively, our work demonstrates the existence of LREs occupied by various key TFs, regulating specific gene expression programs triggered by divergent macrophage polarizing stimuli.

Retinoid X receptor (RXR) is an obligate heterodimeric partner of several nuclear receptors (NRs), and as such a central component of NR signaling regulating the immune and metabolic phenotype of macrophages. Importantly, the binding motifs of RXR heterodimers are enriched in the tissue-selective open chromatin regions of resident macrophages, suggesting roles in subtype specification. Recent genome-wide studies revealed that RXR binds to thousands of sites in the genome, but the mechanistic details how the cistrome is established and serves ligand-induced transcriptional activity remained elusive. Here we show that IL-4-mediated macrophage plasticity results in a greatly extended RXR cistrome via both direct and indirect actions of the transcription factor STAT6. Activation of STAT6 leads to chromatin remodeling and RXR recruitment to de novo enhancers. In addition, STAT6 triggers a secondary transcription factor wave, including PPARγ. PPARγ appears to be indispensable for the development of RXR-bound de novo enhancers, whose activities can be modulated by the ligands of the PPARγ:RXR heterodimer conferring ligand selective cellular responses. Collectively, these data reveal the mechanisms leading to the dynamic extension of the RXR cistrome and identify the lipid-sensing enhancer sets responsible for the appearance of ligand-preferred gene signatures in alternatively polarized macrophages.

MicroRNAs are important components of the post-transcriptional fine-tuning of macrophage gene expression in physiological and pathological conditions. However, the mechanistic underpinnings and the cis-acting genomic factors of how macrophage polarizing signals induce miRNA expression changes are not well characterized. Therefore, we systematically evaluated the transcriptional basis underlying the inflammation-mediated regulation of macrophage microRNome using the combination of different next generation sequencing datasets. We investigated the LPS-induced expression changes at mature miRNA and pri-miRNA levels in mouse macrophages utilizing a small RNA-seq method and publicly available GRO-seq dataset, respectively. Next, we identified an enhancer set associated with LPS-responsive pri-miRNAs based on publicly available H3K4 mono-methylation-specific ChIP-seq and GRO-seq datasets. This enhancer set was further characterized by the combination of publicly available ChIP and ATAC-seq datasets. Finally, direct interactions between the miR-155-coding genomic region and its distal regulatory elements were identified using a 3C-seq approach. Our analysis revealed 15 robustly LPS-regulated miRNAs at the transcriptional level. In addition, we found that these miRNA genes are associated with an inflammation-responsive enhancer network. Based on NFκB-p65 and JunB transcription factor binding, we showed two distinct enhancer subsets associated with LPS-activated miRNAs that possess distinct epigenetic characteristics and LPS-responsiveness. Finally, our 3C-seq analysis revealed the LPS-induced extensive reorganization of the pri-miR-155-associated functional chromatin domain as well as chromatin loop formation between LPS-responsive enhancers and the promoter region. Our genomic approach successfully combines various genome-wide datasets and allows the identification of the putative regulatory elements controlling miRNA expression in classically activated macrophages.

The in situ phenotypic switch of macrophages is delayed in acute injury following irradiation. The combination of bone marrow transplantation and local muscle radiation protection allows for the identification of a myeloid cell contribution to tissue repair. PET-MRI allows monitoring of myeloid cell invasion and metabolism. Altered cellular composition prior to acute sterile injury affects the in situ phenotypic transition of invading myeloid cells to repair macrophages. There is reciprocal intercellular communication between local muscle cell compartments, such as PAX7 positive cells, and recruited macrophages during skeletal muscle regeneration.

Prior exposure to microenvironmental signals could fundamentally change the response of macrophages to subsequent stimuli. It is believed that T helper-2 (Th2)-cell-type cytokine interleukin-4 (IL-4) and Toll-like receptor (TLR) ligand-activated transcriptional programs mutually antagonize each other, and no remarkable convergence has been identified between them. In contrast, here, we show that IL-4-polarized macrophages established a hyperinflammatory gene expression program upon lipopolysaccharide (LPS) exposure. This phenomenon, which we termed extended synergy, was supported by IL-4-directed epigenomic remodeling, LPS-activated NF-κB-p65 cistrome expansion, and increased enhancer activity. The EGR2 transcription factor contributed to the extended synergy in a macrophage-subtype-specific manner. Consequently, the previously alternatively polarized macrophages produced increased amounts of immune-modulatory factors both in vitro and in vivo in a murine Th2 cell-type airway inflammation model upon LPS exposure. Our findings establish that IL-4-induced epigenetic reprogramming is responsible for the development of inflammatory hyperresponsiveness to TLR activation and contributes to lung pathologies.

Gross chromosomal rearrangements (GCRs), including translocations, inversions amplifications, and deletions, can be causal events leading to malignant transformation. GCRs are thought to be triggered by DNA double strand breaks (DSBs), which in turn can be spontaneous or induced by external agents (eg. cytotoxic chemotherapy, ionizing radiation). It has been shown that induction of DNA DSBs at two defined loci can produce stable balanced chromosomal translocations, however, a single engineered DNA DSB could not. Herein, we report that although a single engineered DNA DSB in H2AX "knockdown" cells did not generate GCRs, repair of a single engineered DNA DSB in fibroblasts that had ablated H2ax did produce clonal, stable GCRs, including balanced translocations and megabase-pair inversions. Upon correction of the H2ax deficiency, cells no longer generated GCRs following a single engineered DNA DSB. These findings demonstrate that clonal, stable GCRs can be produced by a single engineered DNA DSB in H2ax knockout cells, and that the production of these GCRs is ameliorated by H2ax expression.

Chronic inflammatory diseases including inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis), psoriasis and rheumatoid arthritis (RA) afflict millions of people worldwide, but their pathogenesis is still not well understood. It is also not well known if distinct changes in gene expression characterize these diseases and if these patterns can discriminate between diseased and control patients and/or stratify the disease. The main focus of our work was the identification of novel markers that overlap among the 3 diseases or discriminate them from each other.

Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.

Multifunctional living materials are attractive due to their powerful ability to self-repair and replicate. However, most natural materials lack electronic functionality. Here we show that an electric field, applied to electricity-producing Geobacter sulfurreducens biofilms, stimulates production of cytochrome OmcZ nanowires with 1,000-fold higher conductivity (30 S cm-1) and threefold higher stiffness (1.5 GPa) than the cytochrome OmcS nanowires that are important in natural environments. Using chemical imaging-based multimodal nanospectroscopy, we correlate protein structure with function and observe pH-induced conformational switching to β-sheets in individual nanowires, which increases their stiffness and conductivity by 100-fold due to enhanced π-stacking of heme groups; this was further confirmed by computational modeling and bulk spectroscopic studies. These nanowires can transduce mechanical and chemical stimuli into electrical signals to perform sensing, synthesis and energy production. These findings of biologically produced, highly conductive protein nanowires may help to guide the development of seamless, bidirectional interfaces between biological and electronic systems.