Granulocyte-macrophage Colony-stimulating Factor Expression Research Articles

Role of GM-CSF in a mouse model of experimental autoimmune prostatitis.

The etiology of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is still unknown. Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to play an important role in the development of autoimmune and inflammatory diseases. Here, we investigated the expression and function of GM-CSF in patients with CP/CPPS and in a mouse model of experimental autoimmune prostatitis (EAP). GM-CSF mRNA levels were detected in expressed prostatic secretions samples from patients with CP/CPPS and in prostate tissue from a mouse model of EAP. The expression of GM-CSF receptor in mouse prostate and dorsal root ganglia were determined using PCR and immunohistochemistry. Behavioral testing and inflammation scoring were performed to evaluate the role of GM-CSF in disease development and symptom severity of EAP using GM-CSF knockout mice. mRNA levels of putative nociceptive and inflammatory markers were measured in the prostate after the induction of EAP. Elevated GM-CSF mRNA levels were observed in expressed prostatic secretions samples from patients with CP/CPPS compared with healthy volunteers. GM-CSF mRNA was also significantly increased in prostate tissue of the EAP mice model. The expression of GM-CSF receptors was confirmed in mouse prostate and dorsal root ganglia. GM-CSF knockout mice showed fewer Infiltrating leukocytes and pain symptoms after the induction of EAP. Deletion of GM-CSF significantly diminished EAP-induced increases of chemokine (C-C motif) ligand 2, chemokine (C-C motif) ligand 3, and nerve growth factor mRNA expression. The results indicated that GM-CSF plays a functional role in the pathogenesis of EAP. GM-CSF may function as a signaling mediator for both inflammation and pain transduction in CP/CPPS.

Block of Granulocyte-Macrophage Colony-Stimulating Factor Prevents Inflammation-Induced Preterm Birth in a Mouse Model for Parturition

A multitude of factors promotes inflammation in the reproductive tract leading to preterm birth. Macrophages peak in the cervix prior to birth and their numbers are increased by the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). We hypothesize GM-CSF is produced from multiple sites in the genital tract and is a key mediator in preterm birth. Ectocervical, endocervical, and amniotic fluid mesenchymal stem cells were treated with lipopolysaccharide (LPS), and the concentration and expression of GM-CSF was measured. Pregnant CD-1 mice on gestational day 17 received LPS and an intravenous injection of either anti-mouse GM-CSF or control antibody. After 6 hours, the preterm birth rate was recorded. Treatment with LPS increased the GM-CSF concentration and messenger RNA expression after 24 hours in all 3 cell lines ( P < .01). Mice treated with LPS and the GM-CSF antibody had a preterm birth rate of 25%, compared to a 66.7% preterm birth rate in controls, within 6 hours ( P < .05, χ2). Treatment with the anti-mouse GM-CSF antibody decreased the concentration of GM-CSF in the mouse serum ( P < .01) but did not alter the number of macrophages or collagen content in the cervix. These studies demonstrate that GM-CSF is produced from multiple sites in the genital tract and that treatment with an antibody to GM-CSF prevents preterm birth. Curiously, the anti-mouse GM-CSF antibody did not decrease the number of macrophages in the cervix. Further research is needed to determine whether antibodies to GM-CSF can be utilized as a therapeutic agent to prevent preterm birth.

Effect of Multiple Vaccinations with Tumor Cell-Based Vaccine with Codon-Modified GM-CSF on Tumor Growth in a Mouse Model.

Ectopic expression of codon-modified granulocyte-macrophage colony-stimulating factor (cGM-CSF) in TC-1 cells (TC-1/cGM-CSF), a model cell line for human papillomavirus (HPV)-infected cervical cancer cells, increased the expression level of GM-CSF and improved the efficacy of tumor cell-based vaccines in a cervical cancer mouse model. The number of vaccine doses required to induce a long-term immune response in a cervical cancer mouse model is poorly understood. Here, we investigated one, three, and five doses of the irradiated TC-1/cGM-CSF vaccine to determine which dose was effective in inducing a greater immune response and the suppression of tumors. Our findings showed that three doses of irradiated TC-1/cGM-CSF vaccine elicited slower tumor growth rates and enhanced survival rates compared with one dose or five doses of irradiated TC-1/cGM-CSF vaccine. Consistently, mice vaccinated with three doses of irradiated TC-1/cGM-CSF vaccine exhibited stronger interferon gamma (IFN-γ) production in HPV E7-specific CD8+ T cells and CD4+ T cells. A higher percentage of natural killer cells and interferon-producing killer dendritic cells (IKDCs) appeared in the splenocytes of the mice vaccinated with three doses of irradiated TC-1/cGM-CSF vaccine compared with those of the mice vaccinated with one dose or five doses of irradiated TC-1/cGM-CSF vaccine. Our findings demonstrate that single or multiple vaccinations, such as five doses, with irradiated TC-1/cGM-CSF vaccine suppressed the immune response, whereas three doses of irradiated TC-1/cGM-CSF vaccine elicited a greater immune response and subsequent tumor suppression.

Tough and Immunosuppressive Titanium-Infiltrated Exoskeleton Matrices for Long-Term Endoskeleton Repair.

Although biodegradable membranes are essential for effective bone repair, severe loss of mechanical stability because of rapid biodegradation, soft tissue invasion, and excessive immune response remain intrinsically problematic. Inspired by the exoskeleton-reinforcing strategy found in nature, we have produced a Ti-infiltrated chitin nanofibrous membrane. The membrane employs vapor-phase infiltration of metals, which often occurs during metal oxide atomic layer deposition (ALD) on organic substrates. This metal infiltration manifests anomalous mechanical improvement and stable integration with chitin without cytotoxicity and immunogenicity. The membrane exhibits both impressive toughness (∼13.3 MJ·m-3) and high tensile strength (∼55.6 MPa), properties that are often mutually exclusive. More importantly, the membrane demonstrates notably enhanced resistance to biodegradation, remaining intact over the course of 12 weeks. It exhibits excellent osteointegrative performance and suppresses the immune response to pathogen-associated molecular pattern molecules indicated by IL-1β, IL-6, and granulocyte-macrophage colony-stimulating factor expression. We believe the excellent chemico-biological properties achieved with ALD treatment can provide insight for synergistic utilization of the polymers and ALD in medical applications.

FGL2 promotes tumor progression in the CNS by suppressing CD103+ dendritic cell differentiation

Few studies implicate immunoregulatory gene expression in tumor cells in arbitrating brain tumor progression. Here we show that fibrinogen-like protein 2 (FGL2) is highly expressed in glioma stem cells and primary glioblastoma (GBM) cells. FGL2 knockout in tumor cells did not affect tumor-cell proliferation in vitro or tumor progression in immunodeficient mice but completely impaired GBM progression in immune-competent mice. This impairment was reversed in mice with a defect in dendritic cells (DCs) or CD103+ DC differentiation in the brain and in tumor-draining lymph nodes. The presence of FGL2 in tumor cells inhibited granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CD103+ DC differentiation by suppressing NF-κB, STAT1/5, and p38 activation. These findings are relevant to GBM patients because a low level of FGL2 expression with concurrent high GM-CSF expression is associated with higher CD8B expression and longer survival. These data provide a rationale for therapeutic inhibition of FGL2 in brain tumors.

Cloning, Optimization of Periplasmic Expression and Purification of Recombinant Granulocyte Macrophage-Stimulating Factor in Escherichia coli BL21 (DE3).

Background:Molgramostim, a nonglycosylated version of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), can be produced in a high level by Escherichia coli. However, overexpression of GM-CSF in bacterial cells usually leads to formation of inclusion bodies and insoluble protein aggregates which are not biologically active. The aim of the present study was to improve the expression of soluble and biologically active GM-CSF in periplasmic space of E. coli BL21 (DE3).Materials and Methods:The codon-optimized GM-CSF gene was subcloned into pET-22b expression vector, in frame with the pelB secretion signal peptide for periplasmic secretion. Cultivation conditions including as isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, incubation temperature, and presence of sucrose were optimized to improve periplasmic expression of GM-CSF. The expressed protein was purified using Ni-NTA affinity column. Biological activity of GM-CSF on HL-60 cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Results:The amount of soluble protein for periplasmic expression was more when compared with one of the cytoplasmic expressions. The optimum condition for periplasmic expression of GM-CSF was expression at 23°C, using 1 mM IPTG as inducer and in the presence of 0.4 M sucrose. The biological activity of purified GM-CSF on HL-60 cell line was assessed by MTT assay, and the specific activity of produced GM-CSF was determined as 1.2 × 104 IU/μg.Conclusion:The present work suggests that periplasmic expression and optimization of cultivation conditions could improve soluble expression of recombinant proteins by E. coli.

High-yield Production of Granulocyte-macrophage Colony-stimulating Factor in E. coli BL21 (DE3) By an Auto-induction Strategy.

A novel strategy to increase protein expression yield is unintended induction of expression in complex media, called auto-induction. This method can be used to express proteins under control of the lac promoter without any need to monitor bacterial growth pattern, and addition of specific expression inducers such as Isopropyl β-D-1-thiogalactopyranoside (IPTG) at proper time. In the present study, a codon optimized gene encoding granulocyte-macrophage colony stimulating factor (GM-CSF) was cloned and over-expressed in Escherichia coli BL21 (DE3) using both conventional inducer-based and auto-induction approaches in a shake flask scale and the yield of GM-CSF expression and biomass production was identified. Results showed higher biomass production and expression yield for GM-CSF in case of auto-induction comparing with IPTG-induction. The auto-induction approach was also performed in a fed batch fermentation process in a 2-L bioreactor scale. The feeding strategy yielded an amount of 300 mg/L GM-CSF within 20 h of induction. However, most of the over-expressed GM-CSF was produced as inclusion bodies and following purification and refolding, a final yield of 90 mg/L was achieved. These results suggest that auto-induction approach can be effectively applied in fed-batch fermentation for the large scale production of GM-CSF; however, further optimization of purification process is obligatory to increase the purification yield.

Inhibitory mechanism of ginsenoside Rh3 on granulocyte–macrophage colony-stimulating factor expression in UV-B–irradiated murine SP-1 keratinocytes

BackgroundUltraviolet (UV) goes through the epidermis and promotes release of inflammatory cytokines in keratinocytes. Granulocyte–macrophage colony-stimulating factor (GM-CSF), one of the keratinocyte-derived cytokines, regulates proliferation and differentiation of melanocytes. Extracellular signal–regulated kinase (ERK1/2) and protein kinase C (PKC) signaling pathways regulate expression of GM-CSF. Based on these results, we found that ginsenoside Rh3 prevented GM-CSF production and release in UV-B–exposed SP-1 keratinocytes and that this inhibitory effect resulted from the reduction of PKCδ and ERK phosphorylation. MethodsWe investigated the mechanism by which ginsenoside Rh3 from Panax ginseng inhibited GM-CSF release from UV-B–irradiated keratinocytes. ResultsTreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or UV-B induced release of GM-CSF in the SP-1 keratinocytes. To elucidate whether the change in GM-CSF expression could be related to PKC signaling, the cells were pretreated with H7, an inhibitor of PKC, and irradiated with UV-B. GM-CSF was decreased by H7 in a dose-dependent manner. When we analyzed which ginsenosides repressed GM-CSF expression among 15 ginsenosides, ginsenoside Rh3 showed the largest decline to 40% of GM-CSF expression in enzyme-linked immunosorbent assay. Western blot analysis showed that TPA enhanced the phosphorylation of PKCδ and ERK in the keratinocytes. When we examined the effect of ginsenoside Rh3, we identified that ginsenoside Rh3 inhibited the TPA-induced phosphorylation levels of PKCδ and ERK. ConclusionIn summary, we found that ginsenoside Rh3 impeded UV-B–induced GM-CSF production through repression of PKCδ and ERK phosphorylation in SP-1 keratinocytes.

Granulocyte-Macrophage Colony-Stimulating Factor as a Therapeutic Target in Multiple Sclerosis

Multiple sclerosis is an inflammatory neurodegenerative disease of the central nervous system (CNS) and the most frequent cause of non-traumatic disability in adults in the Western world. Currently, several drugs have been approved for the treatment of multiple sclerosis. While the newer drugs are more effective, they have less favourable safety profiles. Thus, there is a need to identify new targets for effective and safe therapies, particularly in patients with progressive disease for whom no treatments are available. One such target is granulocyte-macrophage colony-stimulating factor (GM-CSF) or its receptor. In this article we review data on the potential role of GM-CSF and GM-CSF inhibition in MS. We discuss the expression and function of GM-CSF and its receptor in the CNS, as well as data from animal studies and clinical trials in MS.

33 - S-glutathionylation of pyruvate kinase M2 is associated with metabolic reprogramming and cytokine production in the development of allergic obese airway disease

Early on-set allergic obese asthma is an important global clinical problem due to the mounting number of patients and their disease severity that is poorly controlled with available therapeutics. Alterations in metabolism and accompanying changes in redox homeostasis are known to contribute to obesity-associated diseases. Protein S-glutathionylation (PSSG) is a redox-based modification of protein cysteines involving the conjugation of glutathione to the reactive thiol group. We have discovered a direct correlation between PSSG chemistry, glycolysis and inflammation in the lungs from mice fed a high fat diet for 15 weeks. Analysis of lung tissue from obese mice identified that the glycolytic enzyme pyruvate kinase M2 (PKM2), known to catalyze the final step in glycolysis, is a target of s-glutathionylation in the lung tissue from obese mice. Furthermore, we have identified that oxidative stress and s-glutathionylation of PKM2 are increased in primary tracheal epithelial cells cultured in high glucose (25 mM) and treated with palmitate compared to MTECs cultured in normal glucose (5.6 mM). The objective of this study was to identify the s-glutathionylated proteome and investigate if s-glutathionylation of PKM2 alters its protein function to enhance inflammation in the lung tissue from mice treated with house dust mite and subjected to diet induced obesity, a model of early on-set allergic obese asthma. Here we show that s-glutathionylation of PKM2 decreases its glycolytic enzyme activity. This correlates with the upregulation of the inflammatory cytokines thymic stromal lymphopoietin (TSLP) and granulocyte-macrophage colony-stimulating factor (GM-CSF), known to contribute to steroid resistance and neutrophil infiltration in the lung during obesity. Treatment of primary mouse tracheal epithelial cells with the PKM2 small molecule activator TEPP46 attenuates mRNA expression and secretion of TSLP and GM-CSF. Overall, these findings suggest that unique redox perturbations and glycolysis may contribute to the enigmatic endotype of allergic obese asthma.

Immunomodulatory properties of low-dose ionizing radiation on human endothelial cells

Purpose: The application of radiation therapy (RT) is not only used to treat cancer, in Germany, it is also an accepted and empirically established treatment of patients with benign diseases at low doses. The immune modulatory response generated by low-dose RT has a supporting anti-inflammatory effect within the treatment of inflammation-related diseases. The aim of this study was to investigate the effect of ionizing radiation (IR) on the expression and secretion of inflammatory mediators by endothelial cells (ECs) exposed to low and moderate doses.Methods: Non-activated and activated EC were irradiated with doses between 0.01 Gy and 2 Gy with X-rays. Using a multiplex-assay, protein values of interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (G-CSF) and platelet-derived growth factor (PDGF-BB) were measured in the supernatant at different time-points. To investigate possible differences between mRNA expression and protein secretion after IR, the mRNA expression of IL-8, G-CSF and PDGF-BB was determined by real-time quantitative PCR.Results: Radiation treatment caused non-linear dose dependent effects on pro-inflammatory cytokine secretion of IL-8; G-CSF and PDGF-BB. The mRNA-expression levels of those cytokines were non-linear dose-dependent and differed from protein level in the culture supernatant.Conclusions: This study provides deeper insights into the radiobiological effects of radiation doses below 0.3 Gy, in particular 0.05 Gy, and their significant immunomodulatory properties on EC, which is very important in order to assess the effect of LD-IR on EC.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells.

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)γ or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFNα or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFNα, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

Colony-stimulating factors detected in tumor cells and voided urine are potential prognostic markers for patients with muscle-invasive bladder cancer undergoing radical cystectomy.

BackgroundThe clinical use of macrophage colony-stimulating factor, granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) has improved the safety of cytotoxic chemotherapy. However, the overexpression of these CSFs in cancers has been reported to be associated with a poor prognosis in various malignancies. We evaluated the potential of CSF expression as a predictor of clinical outcome in patients with muscle-invasive bladder cancer (MIBC).MethodsConsecutive patients (n=58) with MIBC who underwent radical cystectomy (RC) were included in this retrospective study. Treatment-naïve tumor specimens obtained by initial transurethral resection of bladder tumors prior to RC were immunostained with antibodies against macrophage colony-stimulating factor, G-CSF, and GM-CSF. We compared the clinicopathological variables and survival between these groups. Baseline levels of CSFs in the serum and voided urine were quantified using an enzyme-linked immunosorbent assay and compared with the expression of CSFs in the tumor lesions.ResultsLow expression of GM-CSF in the tumor cells was significantly correlated with a pathological T4 category (vs T2–3; P=0.02). In univariate survival analysis, high G-CSF and low GM-CSF expression in the tumor lesion were associated with poor outcomes. Furthermore, Cox proportional regression analysis revealed that high G-CSF and low GM-CSF expression in the tumor were independent predictors of shorter recurrence-free survival, cancer-specific survival, and overall survival. The levels of CSFs in voided urine were associated with the expression of CSFs in the tumor lesions.ConclusionGM-CSF and G-CSF expression in the tumor lesions obtained by initial transurethral resection are independent predictors of poor outcome in MIBC after RC. Levels of G-CSF and GM-CSF in urine before treatment could be useful in prognostication.

The role of granulocyte macrophage colony stimulating factor in hospitalized children with Mycoplasma pneumoniae pneumonia.

Inappropriate inflammatory response in children with M. pneumoniae infection might be associated with disease severity. The role of Granulocyte macrophage colony stimulating factor (GM-CSF) in hospitalized children with Mycoplasma pneumoniae pneumonia (MPP) has not been fully discussed. Clinical and laboratory data of a total 40 children with MPP were collected. GM-CSF and myeloperoxidase (MPO) were detected by ELISAs. Meanwhile, normal human bronchial epithelium was infected by M.pneumoniae and neutrophils were stimulated by GM-CSF to explore GM-CSF and MPO release in supernatant, respectively. Compared to control group, a significant increased percentage of neutrophils and decreased percentage of macrophages in bronchoalveolar lavage fluid of children with MPP was observed (P<0.05). Children with MPP had significantly higher levels of GM-CSF (P=0.0047) and MPO (P=0.0002) in BALF compared to the controls. Level of GM-CSF in BALF was associated with duration of fever (r=0.42, P=0.007) and strongly correlated with level of MPO (r=0.075, P=0.0005). Levels of GM-CSF and MPO significantly decreased (both P<0.05) after treatment. Invitro, M.pneumoniae induced GM-CSF expression in a time-dependent manner during a 72-h period (P<0.05) and MPO secretion significantly increased by recombinant human GM-CSF stimulation at 24h (P<0.05). GM-CSF could be induced by M.pneumoniae infection in vivo and vitro. Childen with high level GM-CSF had longer duration of fever. GM-CSF probably plays a vital role in neutrophil inflammation in M.pneumoniae infection.

Aerobic Glycolysis Controls Myeloid-Derived Suppressor Cells and Tumor Immunity via a Specific CEBPB Isoform in Triple-Negative Breast Cancer

Myeloid-derived suppressor cells (MDSCs) inhibit anti-tumor immunity. Aerobic glycolysis is a hallmark of cancer. However, the link between MDSCs and glycolysis is unknown in patients with triple-negative breast cancer (TNBC). Here, we detect abundant glycolytic activities in human TNBC. In two TNBC mouse models, 4T1 and Py8119, glycolysis restriction inhibits tumor granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulatingfactor (GM-CSF) expression and reduces MDSCs. These are accompanied with enhanced Tcell immunity, reduced tumor growth and metastasis, and prolonged mouse survival. Mechanistically, glycolysis restriction represses the expression of a specific CCAAT/enhancer-binding protein beta (CEBPB) isoform, liver-enriched activator protein (LAP), via the AMP-activated protein kinase (AMPK)-ULK1 and autophagy pathways, whereas LAP controls G-CSF and GM-CSF expression to support MDSC development. Glycolytic signatures that include lactate dehydrogenase A correlate with high MDSCs and low Tcells, and are associated with poor human TNBC outcome. Collectively, tumor glycolysis orchestrates a molecular network of the AMPK-ULK1, autophagy, and CEBPB pathways to affect MDSCs and maintain tumor immunosuppression.

Endoplasmic reticulum stress in perivascular adipose tissue promotes destabilization of atherosclerotic plaque by regulating GM-CSF paracrine

BackgroundPerivascular adipose tissue (PVAT) accelerates plaque progression and increases cardiovascular risk. We tested the hypothesis that PVAT contributed to plaque vulnerability and investigated whether endoplasmic reticulum stress (ER stress) in PVAT played an important role in vulnerable plaque.MethodsWe transplanted thoracic aortic PVAT or subcutaneous adipose tissue as a control, from donor mice to carotid arteries of recipient apolipoprotein E deficient (apoE−/−) mice after removing carotid artery collar placed for 6 weeks. Two weeks after transplantation, ER stress inhibitor 4-phenyl butyric acid (4-PBA) was locally administrated to the transplanted PVAT and then animals were euthanized after 4 weeks. Immunohistochemistry was performed to quantify plaque composition and neovascularization. Mouse angiogenesis antibody array kit was used to test the angiogenic factors produced by transplanted adipose tissue. In vitro tube formation assay, scratch wound migration assay and mouse aortic ring assay were used to assess the angiogenic capacity of supernatant of transplanted PVAT.ResultsUltrastructural detection by transmission electron microscopy showed transplanted PVAT was a mixed population of white and brown adipocytes with abundant mitochondria. Transplanted PVAT increased the intraplaque macrophage infiltration, lipid core, intimal and vasa vasorum neovascularization and MMP2/9 expression in plaque while decreased smooth muscle cells and collagen in atherosclerotic plaque, which were restored by local 4-PBA-treatment. Antibody array analysis showed that 4-PBA reduced several angiogenic factors [Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), MCP-1, IL-6] secreted by PVAT. Besides, conditioned medium from 4-PBA treated-PVAT inhibited tube formation and migration capacity of endothelial cells and ex vivo mouse aortic ring angiogenesis compared to conditioned medium from transplanted PVAT. mRNA expression and protein levels of GM-CSF were markedly elevated in adipocytes under ER stress which would be suppressed by 4-PBA. In addition, ER stress enhanced NF-κB binding to the promoter of the mouse GM-CSF gene in adipocytes confirmed by Chromatin immunoprecipitation analyses.ConclusionsOur findings demonstrate that ER stress in PVAT destabilizes atherosclerotic plaque, in part through increasing GM-CSF paracrine via transcription factor NF-κB.

Elevated expression of granulocyte-macrophage colony-stimulating factor receptor in multiple sclerosis lesions

Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease that disproportionately affects young adults, leading to disability and high costs to society. Infiltration of T cells and monocytes into the central nervous system (CNS) is critical for disease initiation and progression. However, despite a great deal of effort the molecular mechanisms by which immune cells initiate and perpetuate CNS damage in MS have not yet been elucidated. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by pathogenic Th1 and Th17 cells is critical for the recruitment of monocytes into the CNS during the initial stage of disease. We and others have recently shown that, compared with healthy individuals, MS patients have greater numbers of CD4+ and CD8+ T cells that produce GM-CSF. Here, we describe the expression of GM-CSF and its receptor, GM-CSFR, in normal brain and MS lesions. Our data show that in acute and chronic MS lesions, microglia and astrocytes have upregulated expression of GM-CSFR; in addition, we show that GM-CSF-associated molecules are also upregulated in MS lesions. These findings further strengthen the argument that GM-CSF signaling contributes to MS pathogenesis.

Interaction of RNA-binding protein HuR and miR-466i regulates GM-CSF expression

Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by T helper 17 (Th17) cells plays an essential role in autoimmune diseases. Transcriptional regulation of Th17 cell differentiation has been extensively studied, but post-transcriptional regulation of Th17 cell differentiation has remained less well characterized. The RNA-binding protein HuR functions to promote the stability of target mRNAs via binding the AU-rich elements of the 3′ untranslated region (3′UTR) of numerous pro-inflammatory cytokines including IL-4, IL-13, IL-17 and TNF-α. However, whether HuR regulates GM-CSF expression in Th17 cells has not been fully investigated. Here we showed that HuR conditional knockout (KO) Th17 cells have decreased GM-CSF mRNA in comparison with wild-type (WT) Th17 cells, and that HuR binds directly to GM-CSF mRNA 3′UTR. Interestingly, HuR deficiency increased the levels of certain microRNA expression in Th17 cells; for example, miR-466i functioned to mediate GM-CSF and IL-17 mRNA decay, which was confirmed by in vitro luciferase assay. Furthermore, we found that HuR promoted Mxi1 expression to inhibit certain miRNA expression. Taken together, these findings indicate that interaction of HuR and miR-466i orchestrates GM-CSF expression in Th17 cells.

Effect of the Industrial Nanoparticles TiO 2 , SiO 2 and ZnO on Cell Viability and Gene Expression in Red Bone Marrow of Mus Musculus

To evaluate the effect of ZnO, TiO2 and SiO2 nanoparticles on cell viability and expression of the interleukin 7, interleukin 3, and granulocyte-macrophage colony stimulating factor (GM-CSF) genes in Mus musculus. Red bone marrow was extracted from five Balb/c mice for the analysis of cell viability using the MTT test. The mice were divided into two groups of five each: one group was inoculated intraperitoneally with 0.5, 1.0, 2.5, 5.0, and 10 mg/kg of ZnO and SiO2 nanoparticles, respectively, and the other group was inoculated with 5.0, 10.0, 15.0, 20.0, and 25 mg/kg of TiO2 nanoparticles, respectively. Thirty hours later, RNA was extracted from the red bone marrow of the mice in both groups for gene expression analysis using quantitative PCR and RT-PCR. ZnO and SiO2 nanoparticles reduced cell viability in a dose-dependent manner by 37% and 26%, respectively, starting at a dose of 1 mg/kg. TiO2 nanoparticles at 5 mg/kg and 10 mg/kg reduced the gene expression of interleukins 7 and 3 by 55.3% and 70.2%, respectively, and SiO2 nanoparticles caused the greatest decrease (91%) in the expression of GM-CSF. ZnO nanoparticles reduced the expression of GM-CSF starting at doses of 20 mg/kg and 25 mg/kg. ZnO, SiO2 and TiO2 nanoparticles affect cell viability and gene expression in the mouse bone marrow.

An epithelial-to-mesenchymal transition-inducing potential of granulocyte macrophage colony-stimulating factor in colon cancer

Growing evidence shows that granulocyte macrophage colony-stimulating factor (GM-CSF) has progression-promoting potentials in certain solid tumors, which is largely attributed to the immunomodulatory function of this cytokine in tumor niches. However, little is known about the effect of GM-CSF on cancer cells. Herein, we show that chronic exposure of colon cancer cells to GM-CSF, which harbor its receptor, leads to occurrence of epithelial to mesenchymal transition (EMT), in time and dose-dependent manners. These GM-CSF-educated cancer cells exhibit enhanced ability of motility in vitro and in vivo. Furthermore, GM-CSF stimulation renders colon cancer cells more resistant to cytotoxic agents. Mechanistic investigation reveals that MAPK/ERK signaling and EMT-inducing transcription factor ZEB1 are critical to mediate these effects of GM-CSF. In specimen of CRC patients, high-level expression of GM-CSF positively correlates with local metastases in lymph nodes. Moreover, the co-expression of GM-CSF and its receptors as well as phosphorylated ERK1/2 are observed. Thus, our study for the first time identifies a progression-promoting function of GM-CSF in colon cancer by inducing EMT.