Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1β (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression.

OBJECTIVE

Epidemiological studies have suggested a role of nonalcoholic fatty liver disease (NAFLD) in the development of cardiovascular disease. We evaluated liver mRNA expression of 84 genes encoding proteins involved in the atherosclerosis pathway in patients with NAFLD proven through biopsy in a case-control design, and examined the putative role of the histological disease severity in the molecular events associated with the atherogenic profile.

RESULTS

Nonalcoholic steatohepatitis (NASH), when compared with simple steatosis (SS), significantly increases the expression of TGFB1 (6.8, p<0.005), angiotensin I-converting enzyme (ACE) (2.1, p<0.007), LAMA1 (2.1, p<0.007), SERPINB2 (2.1, p<0.007), CSF2 (2.5, p<0.002), IL1A (2.5, p<0.005), IL3 (2.1, p<0.007), IL4 (2.1, p<0.007), LIF (2.1, p<0.007), and MMP1 (2.1, p<0.007), and decreases the transcript levels of genes involved in the negative regulation of cell-death pathways. A post hoc analysis of liver biopsies of NASH patients who were treated with enalapril monotherapy because of arterial hypertension showed a significant association with lower fibrosis scores in comparison with untreated patients. BIRC3, a severe hypoxia-activated gene, was significantly increased in SS (8.2, p<0.004), when compared with the controls. NASH, but not SS, was also associated with a significant increase in platelet abundance of TGFB1 mRNA. Systems biology analysis revealed highly scored pathways involved in the regulation of programmed cell death, angiogenesis, and immune system, in which TGFB1 was mostly involved.

CONCLUSIONS

NASH, but not SS, may increase atherosclerotic and cardiovascular risk by local overexpression of mediators of atherogenesis, endothelial damage, and regulators of blood pressure; this observation may have therapeutic implications, because ACE inhibitors may improve both cardiovascular outcomes and liver fibrosis. Hepatocyte hypoxia seems to have an important role in the molecular events activated by liver steatosis.

Several compelling lines of evidence suggest an important influence of genetic variation in susceptibility to Kawasaki disease (KD), an acute vasculitis that causes coronary artery aneurysms in children. We performed a family-based genotyping study to test for association between KD and 58 genes involved in cardiovascular disease and inflammation. By analysis of a cohort of 209 KD trios using the transmission disequilibrium test, we documented the asymmetric transmission of five alleles including the interleukin-4 (IL-4) C(-589)T allele (P=0.03). Asymmetric transmission of the IL-4 C(-589)T was replicated in a second, independent cohort of 60 trios (P=0.05, combined P=0.002). Haplotypes of alleles in IL-4, colony-stimulating factor 2 (CSF2), IL-13, and transcription factor 7 (TCF7), all located in the interleukin gene cluster on 5q31, were also asymmetrically transmitted. The reported associations of KD with atopic dermatitis and allergy, elevated serum IgE levels, eosinophilia, and increased circulating numbers of monocyte/macrophages expressing the low-affinity IgE receptor (FCepsilonR2) may be related to effects of IL-4. Thus, the largest family-based genotyping study of KD patients to date suggests that genetic variation in the IL-4 gene, or regions linked to IL-4, plays an important role in KD pathogenesis and disease susceptibility.

The role of mesenchymal stem cells (MSC) in osteosarcoma (OS), the most common primary tumor of bone, has not been extensively elucidated. We have recently shown that OS is characterized by interstitial acidosis, a microenvironmental condition that is similar to a wound setting, in which mesenchymal reactive cells are activated to release mitogenic and chemotactic factors. We therefore intended to test the hypothesis that, in OS, acid-activated MSC influence tumor cell behavior. Conditioned media or co-culture with normal MSC previously incubated with short-term acidosis (pH 6.8 for 10 hr, H+ -MSC) enhanced OS clonogenicity and invasion. This effect was mediated by NF-κB pathway activation. In fact, deep-sequencing analysis, confirmed by Real-Time PCR and ELISA, demonstrated that H+ -MSC differentially induced a tissue remodeling phenotype with increased expression of RelA, RelB and NF-κB1, and downstream, of CSF2/GM-CSF, CSF3/G-CSF and BMP2 colony-promoting factors, and of chemokines (CCL5, CXCL5 and CXCL1), and cytokines (IL6 and IL8), with an increased expression of CXCR4. An increased expression of IL6 and IL8 were found only in normal stromal cells, but not in OS cells, and this was confirmed in tumor-associated stromal cells isolated from OS tissue. Finally, H+ -MSC conditioned medium differentially promoted OS stemness (sarcosphere number, stem-associated gene expression), and chemoresistance also via IL6 secretion. Our data support the hypothesis that the acidic OS microenvironment is a key factor for MSC activation, in turn promoting the secretion of paracrine factors that influence tumor behavior, a mechanism that holds the potential for future therapeutic interventions aimed to target OS.

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.

Granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF2) is a cytokine produced in the hematologic compartment that may enhance antitumor immune responses, mainly by activation of dendritic cells. Here, we show that more than one-third of human colorectal tumors exhibit aberrant DNA demethylation of the GM-CSF promoter and overexpress the cytokine. Mouse engraftment experiments with autologous and homologous colon tumors engineered to repress the ectopic secretion of GM-CSF revealed the tumor-secreted GM-CSF to have an immune-associated antitumor effect. Unexpectedly, an immune-independent antitumor effect was observed that depended on the ectopic expression of GM-CSF receptor subunits by tumors. Cancer cells expressing GM-CSF and its receptor did not develop into tumors when autografted into immunocompetent mice. Similarly, 100% of the patients with human colon tumors that overexpressed GM-CSF and its receptor subunits survived at least 5 years after diagnosis. These data suggest that expression of GM-CSF and its receptor subunits by colon tumors may be a useful marker for prognosis as well as for patient stratification in cancer immunotherapy.

BACKGROUND

Recent fate-mapping studies establish that microglia, the resident mononuclear phagocytes of the CNS, are distinct in origin from the bone marrow-derived myeloid lineage. Interferon regulatory factor 8 (IRF8, also known as interferon consensus sequence binding protein) plays essential roles in development and function of the bone marrow-derived myeloid lineage. However, little is known about its roles in microglia.

METHODS

The CNS tissues of IRF8-deficient mice were immunohistochemically analyzed. Pure microglia isolated from wild-type and IRF8-deficient mice were studied in vitro by proliferation, immunocytochemical and phagocytosis assays. Microglial response in vivo was compared between wild-type and IRF8-deficient mice in the cuprizon-induced demyelination model.

RESULTS

Our analysis of IRF8-deficient mice revealed that, in contrast to compromised development of IRF8-deficient bone marrow myeloid lineage cells, development and colonization of microglia are not obviously affected by loss of IRF8. However, IRF8-deficient microglia demonstrate several defective phenotypes. In vivo, IRF8-deficient microglia have fewer elaborated processes with reduced expression of IBA1/AIF1 compared with wild-type microglia, suggesting a defective phenotype. IRF8-deficient microglia are significantly less proliferative in mixed glial cultures than wild-type microglia. Unlike IRF8-deficient bone marrow myeloid progenitors, exogenous macrophage colony stimulating factor (colony stimulating factor 1) (M-CSF (CSF1)) restores their proliferation in mixed glial cultures. In addition, IRF8-deficient microglia exhibit an exaggerated growth response to exogenous granulocyte-macrophage colony stimulating factor (colony stimulating factor 2) (GM-CSF (CSF2)) in the presence of other glial cells. IRF8-deficient microglia also demonstrate altered cytokine expressions in response to interferon-gamma and lipopolysaccharide in vitro. Moreover, the maximum phagocytic capacity of IRF8-deficient microglia is reduced, although their engulfment of zymosan particles is not overtly impaired. Defective scavenging activity of IRF8-deficient microglia was further confirmed in vivo in the cuprizone-induced demyelination model in mice.

CONCLUSIONS

This study is the first to demonstrate the essential contribution of IRF8-mediated transcription to a broad range of microglial phenotype. Microglia are distinct from the bone marrow myeloid lineage with respect to their dependence on IRF8-mediated transcription.

In this study, we tested the role of colony-stimulating factor 2 (CSF2) as one of the regulatory molecules that mediate maternal effects on embryonic development during the preimplantation period. Our objective was to verify effects of CSF2 on blastocyst yield, determine posttransfer survival, and evaluate properties of the blastocyst formed after CSF2 treatment. In vitro, CSF2 increased the percentage of oocytes that became morulae and blastocysts. Blastocysts that were treated with CSF2 tended to have a greater number of inner cell mass cells and had a higher ratio of inner cell mass to trophectoderm cells. There was no effect of CSF2 on the incidence of apoptosis. Treatment with CSF2 from d 5 to 7 after insemination increased embryonic survival as indicated by improved pregnancy rate at d 30-35 of gestation. Moreover, treatment with CSF2 from either d 1-7 or 5-7 after insemination reduced pregnancy loss after d 30-35. Results indicate that treatment with CSF2 can affect embryonic development and enhance embryo competence for posttransfer survival. The fact that treatment with CSF2 during such a narrow window of development altered embryonic function much later in pregnancy suggests that CSF2 may exert epigenetic effects on the developing embryo that result in persistent changes in function during the embryonic and fetal periods of development.

Prostaglandins (PGs), interleukin 1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF alpha) are likely mediators of local inflammatory reactions. We measured PGE2, PGI2, IL-1 beta, and TNF concentrations in paired cerebrospinal fluid (CSF) samples (on admission, CSF1, and 18 to 30 hours later, CSF2) from 80 infants and children with bacterial meningitis. Forty patients received dexamethasone sodium (0.6 mg/kg per day in four intravenous doses) and 40 received an intravenous saline placebo. In CSF1, PGE2, PGI2, IL-1 beta, and TNF were detected in 90%, 56%, 98%, and 71% of specimens with mean (+/- SEM) concentrations of 462 +/- 65, 377 +/- 62, 1266 +/- 242, and 799 +/- 227 pg/mL, respectively. Concentrations of PGE2 correlated significantly with PGI2, IL-1 beta, TNF, and lactate and inversely correlated with glucose concentrations in the first CSF specimens. The PGE2, PGI2, IL-1 beta, and TNF were still detected in 40%, 18%, 97%, and 60%, respectively, of second CSF specimens obtained from placebo-treated patients. Compared with patients who had detectable PGI2 or TNF alpha concentrations in CSF2 specimens, those placebo-treated patients with no detectable PGI2 or TNF alpha activity in CSF2 had a lower incidence of neurological sequelae. Dexamethasone-treated patients had significantly lower PGE2, IL-1 beta, and lactate concentrations and higher glucose concentrations in CSF 18 to 30 hours later, shorter duration of fever, and a lower incidence of neurological sequelae than did placebo-treated patients.

Interferon (IFN)-gamma plays a critical role in murine uterine spiral artery remodeling for successful pregnancy. The effect of IFN-gamma on human uterine microvasculature, however, remains poorly understood. The aim of this study was to identify the genes regulated by IFN-gamma in human uterine microvascular endothelial cells. The effect of IFN-gamma on the gene expression profile in human uterine microvascular endothelial cells was evaluated by cDNA microarray analysis and quantitative real-time reverse transcriptase-polymerase chain reaction for the selected genes of interest. In vivo expression of the protein encoded by some of these genes in human uterine microvascular endothelial cells was evaluated by Western blotting and immunohistochemistry. Treatment with 10 ng/ml IFN-gamma for 4 h induced a significant>> or =2-fold change in 29 genes in pooled human uterine microvascular endothelial cells; a total of 20 genes were up-regulated, whereas nine genes were down-regulated. The genes significantly up-regulated included chemokines (CXCL9, CXCL10, CCL8, IL15RA, and CCL5), enzymes (GBP5, TAP1, CYP27B1, SOD2, MX1, CASP1, and PTGES), and transcription factors (TFAP2C, IRF1, NFE2L3). The genes significantly down-regulated following IFN-gamma treatment included cytokines/cytokine receptors (CSF2, IL1R2, and SPP1), and insulin-like growth factor binding proteins (WISP2 and IGFBP3). The results of the cDNA microarray analysis were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction for the selected 17 genes of interest. The immunoreactivity for the proteins encoded by IL15RA, IFI30, and MX1 was detected in human uterine microvascular endothelial cells in vivo, whereas the immunoreactivity for CCNA1 and NQO1 was not detectable. These results suggest that IFN-gamma regulates the gene expression involved in natural killer cell recruitment, embryo and trophoblast migration, endometrial decidualization, angiogenesis, angiostasis, and anti-viral infection in human uterine microvascular endothelial cells.

T helper 17 (Th17) cells are important mediators of immune responses against extracellular bacteria and fungi, and as such play critical regulatory roles in maintaining mucosal homeostasis. Conversely, Th17 cells and their effector molecules interleukin 17A (IL-17A), IL-17F, interferon (IFN)γ, tumor necrosis factor (TNF)α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are implicated in the pathology of rheumatoid arthritis (RA). Interactions between Th17 cells and other immune cells or stromal cells that are present in the synovial tissue during the earliest phases of the disease, may eventually lead to chronic inflammation, irreversible cartilage degradation and bone erosions. Recent evidence points towards Th17 cell plasticity as an essential contributing process in RA pathology, since Th17 cells are able to adopt a pathogenic phenotype under the influence of environmental, inflammatory and genetic factors. A remarkable feature of this pathogenic Th17 cell phenotype is the high production of GM-CSF and TNFα and the co-appearance of Th1 cell characteristics, such as transcription factor T-box 21 (T-bet) and IFNγ expression. Recently, much progress has been made in unravelling the mechanisms underlying Th17 cell plasticity and pathogenicity. Of interest, many of the environmental and inflammatory factors associated with RA pathology, such as pro-inflammatory mediators and cytokines, microbiome dysbiosis, metabolism and diet, obesity, vitamins, steroids and hormones are linked to the development of pathogenic Th17 cells. Moreover proteins encoded by established genetic risk factors for RA including CCR6, CD226, CSF2, EOMES, ETS1, GATA3, IL2, IL6R, IL23R, IKZF3, IRAK1, IRF4, IRF8, PRKCQ, PRDM1, RBPJ, RUNX1 and TAGAP are directly involved in Th17 cell differentiation and/or function. This review provides a detailed overview of the molecular mechanisms involved in the heterogeneity and pathogenicity of Th17 cells in the context of autoimmune diseases, with a focus on RA. Understanding these mechanisms creates great potential to identify and select novel therapeutic targets which could improve current therapies or lead to development of new treatment strategies in RA.

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6-8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6-8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation.

BACKGROUND

Granulocyte macrophage colony-stimulating factor (GM-CSF, Csf2) is a growth factor for myeloid-lineage cells that has been implicated in the pathogenesis of atherosclerosis and other chronic inflammatory diseases. However, the role of GM-CSF in advanced atherosclerotic plaque progression, the process that gives rise to clinically dangerous plaques, is unknown.

OBJECTIVE

To understand the role of GM-CSF in advanced atherosclerotic plaque progression.

RESULTS

Ldlr(-/-) mice and Csf2(-/-)Ldlr(-/-) mice were fed a Western-type diet for 12 weeks, and then parameters of advanced plaque progression in the aortic root were quantified. Lesions from the GM-CSF-deficient mice showed a substantial decrease in 2 key hallmarks of advanced atherosclerosis, lesional macrophage apoptosis and plaque necrosis, which indicates that GM-CSF promotes plaque progression. Based on a combination of in vitro and in vivo studies, we show that the mechanism involves GM-CSF-mediated production of interleukin-23, which increases apoptosis susceptibility in macrophages by promoting proteasomal degradation of the cell survival protein Bcl-2 (B-cell lymphoma 2) and by increasing oxidative stress.

CONCLUSIONS

In low-density lipoprotein-driven atherosclerosis in mice, GM-CSF promotes advanced plaque progression by increasing macrophage apoptosis susceptibility. This action of GM-CSF is mediated by its interleukin-23-inducing activity rather than its role as a growth factor.

Uterine dendritic cells (DCs) are critical for activating the T cell response mediating maternal immune tolerance of the semiallogeneic fetus. GM-CSF (CSF2), a known regulator of DCs, is synthesized by uterine epithelial cells during induction of tolerance in early pregnancy. To investigate the role of GM-CSF in regulating uterine DCs and macrophages, Csf2-null mutant and wild-type mice were evaluated at estrus, and in the periconceptual and peri-implantation periods. Immunohistochemistry showed no effect of GM-CSF deficiency on numbers of uterine CD11c(+) cells and F4/80(+) macrophages at estrus or on days 0.5 and 3.5 postcoitum, but MHC class II(+) and class A scavenger receptor(+) cells were fewer. Flow cytometry revealed reduced CD80 and CD86 expression by uterine CD11c(+) cells and reduced MHC class II in both CD11c(+) and F4/80(+) cells from GM-CSF-deficient mice. CD80 and CD86 were induced in Csf2(-/-) uterine CD11c(+) cells by culture with GM-CSF. Substantially reduced ability to activate both CD4(+) and CD8(+) T cells in vivo was evident after delivery of OVA Ag by mating with Act-mOVA males or transcervical administration of OVA peptides. This study shows that GM-CSF regulates the efficiency with which uterine DCs and macrophages activate T cells, and it is essential for optimal MHC class II- and class I-mediated indirect presentation of reproductive Ags. Insufficient GM-CSF may impair generation of T cell-mediated immune tolerance at the outset of pregnancy and may contribute to the altered DC profile and dysregulated T cell tolerance evident in infertility, miscarriage, and preeclampsia.

TLRs trigger innate immunity that recognizes conserved motifs of invading pathogens, resulting in cellular activation and release of inflammatory factors. The influence of TLR activation on resistance to HIV-1 infection has not been investigated in HIV-1 exposed seronegative (ESN) individuals. PBMCs isolated from heterosexually ESN individuals were stimulated with agonists specific for TLR3 (poly I:C), TLR4 (LPS), TLR7 (imiquimod), and TLR7/8 (ssRNA40). We evaluated expression of factors involved in TLR signaling cascades, production of downstream effector immune mediators, and TLR-expression in CD4+ and CD14+ cells. Results were compared with those obtained in healthy controls (HCs). ESN individuals showed: 1) comparable percentages of CD14+/TLR4+ and CD4+/TLR8+ CD14+/TLR8+ cells; 2) higher responsiveness to poly I:C, LPS, imiquimod, and ssRNA40 stimulation, associated with significantly increased production of IL-1beta, IL-6, TNF-alpha, and CCL3; 3) augmented expression of mRNA specific for other targets (CCL2, CSF3, CSF2, IL-1alpha, IL-8, IL-10, IL-12, cyclooxygenase 2) demonstrated by broader TLRs pathway expression analyses; and 4) increased MyD88/MyD88s(short) ratio, mainly following TLR7/8 stimulation. We also compared TLR-agonist-stimulated cytokine/chemokine production in CD14+ PBMCs and observed decreased IFN-beta production in ESN individuals compared with HCs upon TLR7/8-agonist stimulation. These data suggest that TLR stimulation in ESN individuals results in a more robust release of immunologic factors that can influence the induction of stronger adaptive antiviral immune responses and might represent a virus-exposure-induced innate immune protective phenotype against HIV-1.

Runx2 is a metastatic transcription factor (TF) increasingly expressed during prostate cancer (PCa) progression. Using PCa cells conditionally expressing Runx2, we previously identified Runx2-regulated genes with known roles in epithelial-mesenchymal transition, invasiveness, angiogenesis, extracellular matrix proteolysis and osteolysis. To map Runx2-occupied regions (R2ORs) in PCa cells, we first analyzed regions predicted to bind Runx2 based on the expression data, and found that recruitment to sites upstream of the KLK2 and CSF2 genes was cyclical over time. Genome-wide ChIP-seq analysis at a time of maximum occupancy at these sites revealed 1603 high-confidence R2ORs, enriched with cognate motifs for RUNX, GATA and ETS TFs. The R2ORs were distributed with little regard to annotated transcription start sites (TSSs), mainly in introns and intergenic regions. Runx2-upregulated genes, however, displayed enrichment for R2ORs within 40 kb of their TSSs. The main annotated functions enriched in 98 Runx2-upregulated genes with nearby R2ORs were related to invasiveness and membrane trafficking/secretion. Indeed, using SDS-PAGE, mass spectrometry and western analyses, we show that Runx2 enhances secretion of several proteins, including fatty acid synthase and metastasis-associated laminins. Thus, combined analysis of Runx2's transcriptome and genomic occupancy in PCa cells lead to defining its novel role in regulating protein secretion.

Runx2, best known for its role in regulating osteoblast-specific gene expression, also plays an increasingly recognized role in prostate and breast cancer metastasis. Using the C4-2B/Rx2(dox) prostate cancer cell line that conditionally expressed Runx2 in response to doxycycline treatment, we identified and characterized G9a, a histone methyltransferase, as a novel regulator for Runx2 activity. G9a function was locus-dependent. Whereas depletion of G9a reduced expression of many Runx2 target genes, including MMP9, CSF2, SDF1, and CST7, expression of others, such as MMP13 and PIP, was enhanced. Physical association between G9a and Runx2 was indicated by co-immunoprecipitation, GST-pulldown, immunofluorescence, and fluorescence recovery after photobleaching (FRAP) assays. Since G9a makes repressive histone methylation marks and is primarily known as a corepressor, we further investigated the mechanism by which G9a functioned as a positive regulator for Runx2 target genes. Transient reporter assays indicated that the histone methyltransferase activity of G9a was not required for transcriptional activation by Runx2. Chromatin immunoprecipitation assays for Runx2 and G9a showed that G9a was recruited to endogenous Runx2 binding sites. We conclude that a subset of cancer-related Runx2 target genes require recruitment of G9a for their expression, but do not depend on its histone methyltransferase activity.

The objective of the present study was to examine the expression of Toll-like receptors (TLRs) by mouse uterine epithelial cells and to determine if stimulation of the expressed TLR induces changes in cytokine and/or chemokine secretion. Using RT-PCR, the expression of TLRs 1-6 by mouse uterine epithelial cells was demonstrated, with TLRs 7-9 expressed only periodically. In the absence of pathogen-associated molecular patterns, polarized uterine epithelial cells constitutively secrete interleukin (IL) 1A, cysteine-cysteine ligand (CCL) 2, IL6, granulocyte-macrophage colony-stimulating factor 2 (CSF2), tumor necrosis factor A (TNFA), CSF3, and IL8 in vitro, with levels of cytokines/chemokines secreted into the apical compartment being significantly greater than those released into the basolateral compartment. When added to the apical surface for 48 h before analysis, the TLR2-agonist Pam3Cys-Ser-(Lys)4 and TLR1/6-agonist peptidoglycan increased epithelial cell apical secretion of IL1A, CCL2, and IL6 and apical/basolateral bidirectional secretion of CSF2, TNFA, CSF3, and IL8 when compared to controls. The TLR3-agonist poly (I:C) significantly increased bidirectional secretion of CCL2, IL6, TNFA, and CSF2 and basolateral secretion of CSF3. Lastly, the TLR4-agonist lipopolysaccharide increased bidirectional secretion CCL2, CSF2, TNFA, CSF3, and IL8 and apical secretion of IL6. These results indicate that mRNAs for Tlr1 through Tlr6 are expressed by uterine epithelial cells and that treatment with specific TLR agonists alters the expression of key chemokines and proinflammatory cytokines that contribute to the defense of the uterus against potential pathogens.

Heart failure is a complex clinical syndrome characterized by insufficient cardiac function. In addition to abnormalities intrinsic to the heart, dysfunction of other organs and dysregulation of systemic factors greatly affect the development and consequences of heart failure. Here we show that the heart and kidneys function cooperatively in generating an adaptive response to cardiac pressure overload. In mice subjected to pressure overload in the heart, sympathetic nerve activation led to activation of renal collecting-duct (CD) epithelial cells. Cell-cell interactions among activated CD cells, tissue macrophages and endothelial cells within the kidney led to secretion of the cytokine CSF2, which in turn stimulated cardiac-resident Ly6Clo macrophages, which are essential for the myocardial adaptive response to pressure overload. The renal response to cardiac pressure overload was disrupted by renal sympathetic denervation, adrenergic β2-receptor blockade or CD-cell-specific deficiency of the transcription factor KLF5. Moreover, we identified amphiregulin as an essential cardioprotective mediator produced by cardiac Ly6Clo macrophages. Our results demonstrate a dynamic interplay between the heart, brain and kidneys that is necessary for adaptation to cardiac stress, and they highlight the homeostatic functions of tissue macrophages and the sympathetic nervous system.

BACKGROUND

Asthma is a chronic respiratory disease that often originates in early childhood. Although candidate gene studies have identified many potential asthma susceptibility genes in adult populations, few have studied associations with immune phenotypes in the first year that might be early clinical markers of asthma.

OBJECTIVE

The aim of this study was to assess the contribution of genetic variation to cytokine response profiles and atopic phenotypes in the first year of life in the Childhood Origin of Asthma cohort.

METHODS

Two hundred seven European American children participating in the Childhood Origin of Asthma study were genotyped for 61 single nucleotide polymorphisms in 35 genes involved in immune regulation. We examined the relationship between these single nucleotide polymorphisms and PHA-induced cytokine (IL-5, IL-10, IL-13, and IFN-gamma) response profiles at birth and at year 1, respiratory syncytial virus-induced wheezing and atopic dermatitis in the first year of life, and total IgE levels, peripheral blood eosinophil counts, and allergic sensitization at age 1 year. The data were analyzed by using censored regression for quantitative measurements and logistic regression for qualitative phenotypes.

RESULTS

The 237Gly allele of the high-affinity IgE receptor beta chain (FCER1B) and a silent substitution in the nitric oxide synthase (NOS)2A gene were associated with reduced IL-13 responses in cord blood (P = .0025 and P = .0062, respectively). A significant gene-gene interaction between FCER1B 237Gly and NOS2A D346D was detected, with individuals carrying the minor allele for both polymorphisms having the lowest cord blood IL-13 levels. Furthermore, the IL13 110Gln allele showed an association with increased IgE levels at year 1 (P = .0026), and the colony-stimulating factor 2 (CSF2) 117Thr allele showed an association with a greater increase in IL-5 responses during the first year (P = .0092). The TGF-beta1 (TGFB1) -509T allele was associated with respiratory syncytial virus-related wheezing in the first year (P = .0005). None of the polymorphisms included in this study were associated with atopic dermatitis during the first year or a positive RAST result at 1 year of age.

CONCLUSIONS

These data suggest that variations in genes involved in immune regulation are associated with biologic and clinical phenotypes in the first year of life that might increase the risk for the subsequent development of childhood asthma.

Mutations in the colony stimulating factor-1 receptor (CSF1R) that abrogate the expression of the affected allele or lead to the expression of mutant receptor chains devoid of kinase activity have been identified in both familial and sporadic cases of ALSP. To determine the validity of the Csf1r heterozygous mouse as a model of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) we performed behavioral, radiologic, histopathologic, ultrastructural and cytokine expression studies of young and old Csf1r+/- and control Csf1r+/+ mice. Six to 8-month old Csf1r+/- mice exhibit cognitive deficits, and by 9-11 months develop sensorimotor deficits and in male mice, depression and anxiety-like behavior. MRIs of one year-old Csf1r+/- mice reveal lateral ventricle enlargement and thinning of the corpus callosum. Ultrastructural analysis of the corpus callosum uncovers dysmyelinated axons as well as neurodegeneration, evidenced by the presence of axonal spheroids. Histopathological examination of 11-week-old mice reveals increased axonal and myelin staining in the cortex, increase of neuronal cell density in layer V and increase of microglial cell densities throughout the brain, suggesting that early developmental changes contribute to disease. By 10-months of age, the neuronal cell density normalizes, oligodendrocyte precursor cells increase in layers II-III and V and microglial densities remain elevated without an increase in astrocytes. Also, the age-dependent increase in CSF-1R+ neurons in cortical layer V is reduced. Moreover, the expression of Csf2, Csf3, Il27 and Il6 family cytokines is increased, consistent with microglia-mediated inflammation. These results demonstrate that the inactivation of one Csf1r allele is sufficient to cause an ALSP-like disease in mice. The Csf1r+/- mouse is a model of ALSP that will allow the critical events for disease development to be determined and permit rapid evaluation of therapeutic approaches. Furthermore, our results suggest that aberrant activation of microglia in Csf1r+/- mice may play a central role in ALSP pathology.

Acute myeloid leukemia (AML) in Down syndrome (DS) children has several unique features including a predominance of the acute megakaryocytic leukemia (AMkL) phenotype, higher event-free survivals compared to non-DS children using cytosine arabinoside (ara-C)/anthracycline-based protocols and a uniform presence of somatic mutations in the X-linked transcription factor gene, GATA1. Several chromosome 21-localized transcription factor oncogenes including ETS2 may contribute to the unique features of DS AMkL. ETS2 transcripts measured by real-time RT-PCR were 1.8- and 4.1-fold, respectively, higher in DS and non-DS megakaryoblasts than those in non-DS myeloblasts. In a doxycycline-inducible erythroleukemia cell line, K562pTet-on/ETS2, induction of ETS2 resulted in an erythroid to megakaryocytic phenotypic switch independent of GATA1 levels. Microarray analysis of doxycycline-induced and doxycycline-uninduced cells revealed an upregulation by ETS2 of cytokines (for example, interleukin 1 and CSF2) and transcription factors (for example, TAL1), which are key regulators of megakaryocytic differentiation. In the K562pTet-on/ETS2 cells, ETS2 induction conferred differences in sensitivities to ara-C and daunorubicin, depending on GATA1 levels. These results suggest that ETS2 expression is linked to the biology of AMkL in both DS and non-DS children, and that ETS2 acts by regulating expression of hematopoietic lineage and transcription factor genes involved in erythropoiesis and megakaryopoiesis, and in chemotherapy sensitivities.

Pathogenic lymphocytes initiate the development of chronic inflammatory diseases. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) (encoded by Csf2) is a key communicator between pathogenic lymphocytes and tissue-invading inflammatory phagocytes. However, the molecular properties of GM-CSF-producing cells and the mode of Csf2 regulation in vivo remain unclear. To systematically study and manipulate GM-CSF⁺ cells and their progeny in vivo, we generated a fate-map and reporter of GM-CSF expression mouse strain (FROG). We mapped the phenotypic and functional profile of auto-aggressive T helper (Th) cells during neuroinflammation and identified the signature and pathogenic memory of a discrete encephalitogenic Th subset. These cells required interleukin-23 receptor (IL-23R) and IL-1R but not IL-6R signaling for their maintenance and pathogenicity. Specific ablation of this subset interrupted the inflammatory cascade, despite the unperturbed tissue accumulation of other Th subsets (e.g., Th1 and Th17), highlighting that GM-CSF expression not only marks pathogenic Th cells, but that this subset mediates immunopathology and tissue destruction.

Genetic deficiency in granulocyte-macrophage colony-stimulating factor (CSF2, GM-CSF) results in altered placental structure in mice. To investigate the mechanism of action of CSF2 in placental morphogenesis, the placental gene expression and cell composition were examined in Csf2 null mutant and wild-type mice. Microarray and quantitative RT-PCR analyses on Embryonic Day (E) 13 placentae revealed that the Csf2 null mutation caused altered expression of 17 genes not previously known to be associated with placental development, including Mid1, Cd24a, Tnfrsf11b, and Wdfy1. Genes controlling trophoblast differentiation (Ascl2, Tcfeb, Itgav, and Socs3) were also differentially expressed. The CSF2 ligand and the CSF2 receptor alpha subunit were predominantly synthesized in the placental junctional zone. Altered placental structure in Csf2 null mice at E15 was characterized by an expanded junctional zone and by increased Cx31(+) glycogen cells and cyclin-dependent kinase inhibitor 1C (CDKN1C(+), P57(Kip2+)) giant cells, accompanied by elevated junctional zone transcription of genes controlling spongiotrophoblast and giant cell differentiation and secretory function (Ascl2, Hand1, Prl3d1, and Prl2c2). Granzyme genes implicated in tissue remodeling and potentially in trophoblast invasion (Gzmc, Gzme, and Gzmf) were downregulated in the junctional zone of Csf2 null mutant placentae. These data demonstrate aberrant placental gene expression in Csf2 null mutant mice that is associated with altered differentiation and/or functional maturation of junctional zone trophoblast lineages, glycogen cells, and giant cells. We conclude that CSF2 is a regulator of trophoblast differentiation and placental development, which potentially influences the functional capacity of the placenta to support optimal fetal growth in pregnancy.