Granulocyte-macrophage Colony-stimulating Factor mRNA Research Articles

Astragalus regulates the intestinal immune response during sepsis by mediating ILC3 proliferation through RORγt

BackgroundSepsis is a common complication of many diseases and is associated with high morbidity and mortality rates. Astragalus can improve humoral and innate immunity, inhibit inflammatory responses, and protect immune cells and organs from damage. However, to the best of our knowledge there are no reports on whether astragalus can regulate intestinal innate immune function during sepsis. MethodsIn this study, a rat cecal ligation and puncture model of sepsis was used to investigate the effects of astragalus treatment, following which the apoptosis rate of lymphocytes from Peyer’s patches (PP) was determined. Type 3 innate lymphoid cells (ILC3) were cultured in vitro to further evaluate the effects and mechanisms of astragalus. ResultsThe apoptosis level of lymphocytes from PP in rats with sepsis was significantly increased, and the number of ILC3 was significantly reduced, compared with the sham operation group, which aggravated intestinal injury and ultimately led to the death of rats. Astragalus treatment significantly inhibited the apoptosis of lymphocytes from PP, increased the number of ILC3, and improved the intestinal inflammatory environment compared to the sepsis group. RT-PCR revealed that astragalus and the retinoic acid-related orphan receptor γt (RORγt) agonist LYC-55716 both promote the expression of interleukin (IL)-17A, IL-17F, IL-22, interferon-γ, and granulocyte-macrophage colony-stimulating factor mRNA. Mechanistically, astragalus promotes the proliferation of ILC3 through RORγt, thereby reducing intestinal inflammatory damage. ConclusionAstragalus, via RORγt, promotes the generation of ILC3, improves the inflammatory environment in rats with sepsis.

Modular Hydrogel Vaccine for Programmable and Coordinate Elicitation of Cancer Immunotherapy.

Immunotherapy holds great promise for the treatment of malignant cancer. However, the lack of sufficient tumor neoantigens and incomplete dendritic cell (DC) maturation compromise the efficacy of immunotherapy. Here, a modular hydrogel-based vaccine capable of eliciting a powerful and sustained immune response is developed. Briefly, CCL21a and ExoGM-CSF+Ce6 (tumor cell-derived exosomes with granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA encapsulated inside and sonosensitizer chlorin e6 (Ce6) incorporated in the surface) are mixed with nanoclay and gelatin methacryloyl, forming the hydrogel designated as CCL21a/ExoGM-CSF+Ce6 @nanoGel. The engineered hydrogel releases CCL21a and GM-CSF with a time gap. The earlier released CCL21a diverts the tumor-draining lymph node (TdLN) metastatic tumor cells to the hydrogel. Consequently, the trapped tumor cells in the hydrogel, in turn, engulf the Ce6-containing exosomes and thus are eradicated by sonodynamic therapy (SDT), serving as the antigen source. Later, together with the remnant CCL21a, GM-CSF produced by cells engulfing ExoGM-CSF+Ce6 continuously recruits and provokes DCs. With the two programmed modules, the engineered modular hydrogel vaccine efficiently inhibits tumor growth and metastasis via diverting TdLN metastatic cancer to hydrogel, killing the trapped tumor cells, and eliciting prolonged and powerful immunotherapy in an orchestrated manner. The strategy would open an avenue for cancer immunotherapy.

Dense GM-CSFRα-expressing immune infiltration is allied with longer survival of intrahepatic cholangiocarcinoma patients.

Intrahepatic cholangiocarcinoma (iCCA) is a cancer arising from intrahepatic bile duct epithelium. An iCCA incidence is increasing worldwide; however, the outcome of the disease is dismal. The linkage between chronic inflammation and iCCA progression is well established, but the roles of granulocyte-macrophage colony-stimulating factor (GM-CSF) remain unrevealed. Thus, a better understanding of GM-CSF functions in CCA may provide an alternative approach to CCA treatment. Differential GM-CSF and GM-CSFRα mRNA expressions in CCA tissues were investigated by Gene Expression Profiling Interactive Analysis (GEPIA) based on The Cancer Genome Atlas (TCGA) database. The protein expressions and localizations of GM-CSF and its cognate receptor (GM-CSFRα) in iCCA patients' tissues were demonstrated by the immunohistochemistry (IHC) techniques. The survival analyses were performed using Kaplan-Meier survival analysis with log-rank test and Cox proportional hazard regression model for multivariate analysis. The GM-CSF productions and GM-CSFRα expressions on CCA cells were assessed by ELISA and flow cytometry. The effects of GM-CSF on CCA cell proliferation and migration were evaluated after recombinant human GM-CSF treatment. The relationship between GM-CSF or GM-CSFRα level and related immune cell infiltration was analyzed using the Tumor Immune Estimation Resource (TIMER). GEPIA analysis indicated GM-CSF and GM-CSFRα expressions were higher in CCA tissues than in normal counterparts, and high GM-CSFRα was related to the longer disease-free survival of the patients (p < 0.001). IHC analysis revealed that CCA cells differentially expressed GM-CSF, while GM-CSFRα was expressed on cancer-infiltrating immune cells. The patient whose CCA tissue contained high GM-CSF expressed CCA, and moderate to dense GM-CSFRα-expressing immune cell infiltration (ICI) acquired longer overall survival (OS) (p = 0.047), whereas light GM-CSFRα-expressing ICI contributed to an increased hazard ratio (HR) to 1.882 (95% CI [1.077-3.287]; p = 0.026). In non-papillary subtype, an aggressive CCA subtype, patients with light GM-CSFRα-expressing ICI had shorter median OS (181 vs. 351 days; p = 0.002) and the HR was elevated to 2.788 (95% CI [1.299-5.985]; p = 0.009). Additionally, TIMER analysis demonstrated GM-CSFRα expression was positively correlated with neutrophil, dendritic cell, and CD8+ T cell infiltrations, though it was conversely related to M2-macrophage and myeloid-derived suppressor cell infiltration. However, the direct effects of GM-CSF on CCA cell proliferation and migration were not observed in the current study. Light GM-CSFRα-expressing ICI was an independent poor prognostic factor for iCCA patients. Anti-cancer functions of GM-CSFRα-expressing ICI were suggested. Altogether, the benefits of acquired GM-CSFRα-expressing ICI and GM-CSF for CCA treatment are proposed herein and require elucidation.

Persistent Infection of a Canine Histiocytic Sarcoma Cell Line with Attenuated Canine Distemper Virus Expressing Vasostatin or Granulocyte-Macrophage Colony-Stimulating Factor.

Canine histiocytic sarcoma (HS) represents a neoplasia with poor prognosis. Due to the high metastatic rate of HS, there is urgency to improve treatment options and to prevent tumor metastases. Canine distemper virus (CDV) is a single-stranded negative-sense RNA (ssRNA (-)) virus with potentially oncolytic properties. Moreover, vasostatin and granulocyte-macrophage colony-stimulating factor (GM-CSF) are attractive molecules in cancer therapy research because of their anti-angiogenetic properties and potential modulation of the tumor microenvironment. In the present study, an in vitro characterization of two genetically engineered viruses based on the CDV strain Onderstepoort (CDV-Ond), CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF was performed. Canine histiocytic sarcoma cells (DH82 cells) were persistently infected with CDV-Ond, CDV-Ondneon, CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF and characterized on a molecular and protein level regarding their vasostatin and GM-CSF production. Interestingly, DH82 cells persistently infected with CDV-Ondneon-vasostatin showed a significantly increased number of vasostatin mRNA transcripts. Similarly, DH82 cells persistently infected with CDV-Ondneon-GM-CSF displayed an increased number of GM-CSF mRNA transcripts mirrored on the protein level as confirmed by immunofluorescence and Western blot. In summary, modified CDV-Ond strains expressed GM-CSF and vasostatin, rendering them promising candidates for the improvement of oncolytic virotherapies, which should be further detailed in future in vivo studies.

LTF Regulates the Immune Microenvironment of Prostate Cancer Through JAK/STAT3 Pathway

BackgroundThe study of the immune microenvironment in prostate cancer (PRAD) has brought new opportunities for the current traditional treatment regimens. Therefore, our goal is to develop a universal immunodiagnostic marker to improve patient survival.MethodsBioinformatics analysis: We collected 591 samples from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts and evaluated the abundance and distribution of immune cell members in the PRAD expression profile matrix in the mixed cell population by CIBERSORT, ESTIMATE, single-sample gene set enrichment analysis (ssGSEA), and other methods. The target genes related to PRAD immune microenvironment and tumor mutation load were obtained by overlap analysis and verified by pan-cancer analysis. Cell experiment: The cell transfection scheme was designed, and the experiment was divided into three groups: overexpressing lactoferrin (LTF) group, empty plasmid group, and control group. After obtaining cells in each group, the gene and protein expression levels of LTF and signal transduction of signal transducer and activator of transcription 3 (STAT3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the above three groups were detected by real-time PCR and Western blot, respectively. Finally, the level of GM-CSF secretion in the three groups was detected by ELISA.ResultsMacrophages, resting mast cells, and plasma cells play an important role in PRAD immune microenvironment. In addition, high tumor mutation load [tumor mutational burden (TMB)] was positively correlated with lymph node metastasis in patients with PRAD. As the core gene of the PRAD immune microenvironment, the low expression of LTF in PRAD promotes the occurrence of immunodeficiency, PRAD, and the enrichment of the Janus kinase (JAK)/STAT3 signal pathway. Through cell experiments, it was found that the content of LTF mRNA and protein increased significantly, while the content of STAT3 and GM-CSF mRNA and protein decreased significantly in the overexpressed LTF group. The level of GM-CSF in the supernatant of cell culture was significantly decreased in the overexpression group of LTF.ConclusionThe core gene we proposed is one of the most promising biomarkers to improve the overall survival rate of PRAD and provides an important theoretical basis for the study of the mechanism of the LTF-mediated JAK/STAT3 pathway in PRAD.

Granulocyte-macrophage colony-stimulating factor mRNA and Neuroprotective Immunity in Parkinson's disease

Restoring numbers and function of regulatory T cells (Tregs) is a novel therapeutic strategy for neurodegenerative disorders. Whether Treg function is boosted by adoptive cell transfer, pharmaceuticals, or immune modulators, the final result is a robust anti-inflammatory and neuronal sparing response. Herein, a newly developed lipid nanoparticle (LNP) containing mRNA encoding granulocyte-macrophage colony-stimulating factor (Gm-csf mRNA) was developed to peripherally induce Tregs and used for treatment in preclinical Parkinson's disease (PD) models. Administration of Gm-csf mRNA to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and rats overexpressing alpha-synuclein produced dose-dependent increases in plasma GM-CSF levels and peripheral CD4+CD25+FoxP3+ Treg populations. This upregulation paralleled nigrostriatal neuroprotection, upregulated immunosuppression-associated mRNAs that led to the detection of a treatment-induced CD4+ T cell population, and decreased reactive microgliosis. The current findings strengthen prior works utilizing immune modulation by harnessing Gm-csf mRNA to augment adaptive immune function by employing a new delivery platform to treat PD and potentially other neurodegenerative disorders.

Knockdown of lncRNA ANRIL suppresses the production of inflammatory cytokines and mucin 5AC in nasal epithelial cells via the miR-15a-5p/JAK2 axis.

The incidence of allergic rhinitis (AR) is increasing worldwide. Human nasal epithelial cells (HNECs) are the key cells in the occurrence of AR. Antisense non-coding RNA in the INK4 locus (ANRIL) was discovered to be involved in the progression of AR. However, the mechanism by which ANRIL mediates the progression of AR remains to be determined. The present study aimed to further explore the mechanism by which ANRIL regulates AR. Thereby, HNECs were treated with IL-13 to mimic AR in vitro. The mRNA expression levels of ANRIL, microRNA (miR)-15a-5p, JAK2, mucin 5AC (MUC5AC), granulocyte-macrophage colony-stimulating factor (GM-CSF) and eotaxin-1, and protein expression levels of JAK2, STAT3 and phosphorylated-STAT3 in HNECs were analyzed using reverse transcription-quantitative PCR and western blotting, respectively. ELISAs were used to detect the secretory levels of inflammatory cytokines and mucin in cell supernatants. In addition, a dual luciferase reporter assay was used to confirm the downstream target of ANRIL and the target gene of miR-15a-5p. The results revealed that the secretory levels of eotaxin-1, GM-CSF and MUC5AC were significantly upregulated by IL-13 in the supernatant of HNECs. The expression levels of ANRIL and JAK2 were also upregulated in IL-13-induced HNECs, while the expression levels of miR-15a-5p were downregulated. In addition, ANRIL was identified to bind to miR-15a-5p. The IL-13-induced upregulation of eotaxin-1, GM-CSF and MUC5AC mRNA expression and secretory levels was significantly inhibited by the genetic knockdown of ANRIL, while the miR-15a-5p inhibitor effectively reversed this effect. JAK2 was also discovered to be directly targeted by miR-15a-5p. The overexpression of JAK2 significantly suppressed the therapeutic effect of miR-15a-5p mimics on IL-13-induced inflammation in vitro. In conclusion, the findings of the present study suggested that the genetic knockdown of ANRIL may suppress the production of inflammatory cytokines and mucin in IL-13-treated HNECs via regulation of the miR-15a-5p/JAK2 axis. Thus, ANRIL may serve as a novel target for AR treatment.

Inhibition of purinergic P2X receptor 7 (P2X7R) decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in U251 glioblastoma cells

Glioblastoma is the most aggressive form of primary brain cancer, with a median survival of 12–15 months. The P2X receptor 7 (P2X7R) is upregulated in glioblastoma and is associated with increased tumor cell proliferation. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is also upregulated in glioblastoma and has been shown to have both pro- and anti-tumor functions. This study investigates the potential mechanism linking P2X7R and GM-CSF in the U251 glioblastoma cell line and the therapeutic potential of P2X7R antagonism in this setting. P2X7R protein and mRNA was demonstrated to be expressed in the U251 cell line as assessed by immunocytochemistry and qPCR. Its channel function was intact as demonstrated by live cell confocal imaging using a calcium indicator Fluo-4 AM. Inhibition of P2X7R using antagonist AZ10606120, decreased both GM-CSF mRNA (P < 0.05) and protein (P < 0.01) measured by qPCR and ELISA respectively. Neutralization of GM-CSF with an anti-GM-CSF antibody did not alter U251 cell proliferation, however, P2X7R antagonism with AZ10606120 significantly reduced U251 glioblastoma cell numbers (P < 0.01). This study describes a novel link between P2X7R activity and GM-CSF expression in a human glioblastoma cell line and highlights the potential therapeutic benefit of P2X7R inhibition with AZ10606120 in glioblastoma.

Generation of a novel CD30+ B cell subset producing GM-CSF and its possible link to the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a T helper type 2 (Th2)-associated autoimmune disease characterized by vasculopathy and fibrosis. Efficacy of B cell depletion therapy underscores antibody-independent functions of B cells in SSc. A recent study showed that the Th2 cytokine interleukin (IL)-4 induces granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing effector B cells (GM-Beffs ) in humans. In this study, we sought to elucidate the generation mechanism of GM-Beffs and also determine a role of this subset in SSc. Among Th-associated cytokines, IL-4 most significantly facilitated the generation of GM-Beffs within memory B cells in healthy controls (HCs). In addition, the profibrotic cytokine transforming growth factor (TGF)-β further potentiated IL-4- and IL-13-induced GM-Beffs . Of note, tofacitinib, a Janus kinase (JAK) inhibitor, inhibited the expression of GM-CSF mRNA and protein in memory B cells induced by IL-4, but not by TGF-β. GM-Beffs were enriched within CD20+ CD30+ CD38-/low cells, a distinct population from plasmablasts, suggesting that GM-Beffs exert antibody-independent functions. GM-Beffs were also enriched in a CD30+ fraction of freshly isolated B cells. GM-Beffs generated under Th2 conditions facilitated the differentiation from CD14+ monocytes to DC-SIGN+ CD1a+ CD14- CD86+ cells, which significantly promoted the proliferation of naive T cells. CD30+ GM-Beffs were more pronounced in patients with SSc than in HCs. A subpopulation of SSc patients with the diffuse type and concomitant interstitial lung disease exhibited high numbers of GM-Beffs . Together, these findings suggest that human GM-Beffs are enriched in a CD30+ B cell subset and play a role in the pathogenesis of SSc.

Increased Pulmonary GM-CSF Causes Alveolar Macrophage Accumulation. Mechanistic Implications for Desquamative Interstitial Pneumonitis.

Desquamative interstitial pneumonia (DIP) is a rare, smoking-related, diffuse parenchymal lung disease characterized by marked accumulation of alveolar macrophages (AMs) and emphysema, without extensive fibrosis or neutrophilic inflammation. Because smoking increases expression of pulmonary GM-CSF (granulocyte/macrophage-colony stimulating factor) and GM-CSF stimulates proliferation and activation of AMs, we hypothesized that chronic exposure of mice to increased pulmonary GM-CSF may recapitulate DIP. Wild-type (WT) mice were subjected to inhaled cigarette smoke exposure for 16 months, and AM numbers and pulmonary GM-CSF mRNA levels were measured. After demonstrating that smoke inhalation increased pulmonary GM-CSF in WT mice, transgenic mice overexpressing pulmonary GM-CSF (SPC-GM-CSF+/+) were used to determine the effects of chronic exposure to increased pulmonary GM-CSF (without smoke inhalation) on accumulation and activation of AMs, pulmonary matrix metalloproteinase (MMP) expression and activity, lung histopathology, development of polycythemia, and survival. In WT mice, smoke exposure markedly increased pulmonary GM-CSF and AM accumulation. In unexposed SPC-GM-CSF+/+ mice, AMs were spontaneously activated as shown by phosphorylation of STAT5 (signal inducer and activator of transcription 5) and accumulated progressively with involvement of 84% (interquartile range, 55-90%) of the lung parenchyma by 10 months of age. Histopathologic features also included scattered multinucleated giant cells, alveolar epithelial cell hyperplasia, and mild alveolar wall thickening. SPC-GM-CSF+/+ mice had increased pulmonary MMP-9 and MMP-12 levels, spontaneously developed emphysema and secondary polycythemia, and had increased mortality compared with WT mice. Results show cigarette smoke increased pulmonary GM-CSF and AM proliferation, and chronically increased pulmonary GM-CSF recapitulated the cardinal features of DIP, including AM accumulation, emphysema, secondary polycythemia, and increased mortality in mice. These observations suggest pulmonary GM-CSF may be involved in the pathogenesis of DIP.

ACSL1 Regulates TNFα-Induced GM-CSF Production by Breast Cancer MDA-MB-231 Cells.

Overexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in different types of cancer is associated with tumor growth and progression. Tumor necrosis factor-α (TNFα) is involved in the induction of GM-CSF in different cells; however, the underlying molecular mechanism in this production of GM-CSF has not been fully revealed. Recently, it was noted that TNFα mediates inflammatory responses through long-chain acyl-CoA synthetase 1 (ACSL1). Therefore, we investigated the role of ACSL1 in the TNFα mediated production of GM-CSF. Our results showed that MDA-MB-231 cells displayed increased GM-CSF mRNA expression and secretion after incubation with TNFα. Blocking of ACSL1 activity in the cells with triacsin C markedly suppressed the secretion of GM-CSF. However, inhibition of β-oxidation and ceramide biosynthesis were not required for GM-CSF production. By small interfering RNA mediated knockdown, we further demonstrated that TNFα induced GM-CSF production was significantly diminished in ACSL1 deficient cells. TNFα mediated GM-CSF expression was significantly reduced by inhibition of p38 MAPK, ERK1/2 and NF-κB signaling pathways. TNFα induced phosphorylation of p38, ERK1/2, and NF-κB was observed during the secretion of GM-CSF. On the other hand, inhibition of ACSL1 activity attenuates TNFα mediated phosphorylation of p38 MAPK, ERK1/2, and NF-κB in the cells. Importantly, our findings suggest that ACSL1 plays an important role in the regulation of GM-CSF induced by TNFα in MDA-MB-231 cells. Therefore, ACSL1 may be considered as a potential novel therapeutic target for tumor growth.

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.

Corrigendum: Effect of Cigarette Smoke Extract on Lipopolysaccharide-Activated Mitogen-Activated Protein Kinase Signal Transduction Pathway in Cultured Cells.

Lipopolysaccharide (LPS) forms outer membrane of the wall of Gram-negative cells. LPS can directly cause damage to epithelia of respiratory tract and is the major factor responsible for the chronic inflammation of respiratory passage. The mitogen-activated protein kinase (MAPK) signal transduction pathway of the airway epithelia is intimately associated with the action of LPS. The chronic inflammation of respiratory tract and smoking are interrelated and entwined in the development and progression of chronic lung diseases. This study was designed to examine the effects of cigarette smoke extract (CSE) and LPS on MAPK signal transduction pathway in order to further understand the roles CSE and LPS play in chronic lung inflammation.Cultured primary human epithelial cells of airway were divided into four groups according to the stimulants used: blank control group, LPS-stimulation group, CSE-stimulation group and CSE plus LPS group. Western blotting was employed for the detection of phosphorylation level of extracellular-signal-regulated-kinase (ERK(1/2)), p38 MAPK and c-Jun N-terminal kinase (JNK). The expression of cytokines of MAPK transduction pathway (granulocyte-macrophage colony stimulating factor (GM-CSF) and mRNA of IL-8) in the primary epithelial cells of respiratory tract was also determined.Western blotting revealed that the phosphorylation levels of ERK(1/2), p38 MAPK and JNK were low and 2 hours after the LPS stimulation, the phosphorylation of ERK(1/2), p38 MAPK and JNK were all increased. There was a significant difference in the phosphorylation between the LPS-stimulation group and blank control group (P < 0.05); no significant difference was found between CSE-stimulation group and blank control group (P > 0.05); there was a significant difference between CSE + LPS group and blank control group and between CSE + LPS group and LPS group (P < 0.05). The phosphorylation of CSE-LPS group was higher than that of blank control group but lower than that of LPS group. In blank control group, the expression of IL-8 and GM-CSF mRNA was low in the epithelial cells of airway and the release of IL-8 and GM-CSF was also at a low level. One hour after LPS stimulation, the level of IL-8 mRNA increased (P < 0.05) and reached a peak after 2 hours. On the other hand, GM-CSF mRNA level increased 2 hours after the stimulation (P < 0.05) and reached the highest level 4 hours after the stimulation. Two hours after LPS stimulation, IL-8 and GM-CSF protein level began to rise (P < 0.05), and the level was the highest 8 hours after the stimulation (P < 0.01). Stimulation with CSE alone had no effect on the release of IL-8 and GM-CSF and expression of IL-8 mRNA (P > 0.05), but pre-treatment with CSE could delay the LPS-induced release of IL-8 and GM-CSF and the expression of IL-8 mRNA and its peak was lower.LPS stimulation can significantly increase the phosphorylation of ERK(1/2), p38 MAPK and JNK in the epithelial cells of airway and activate the MAPK transduction pathway, thereby can activate the downstream signal transduction pathway, and can ultimately result in the release of cytokines by the epithelial cells of airway. CSE can partially abolish the LPS-induced activation of MAPK signal transduction pathway and the expression of cytokines of the pathway, which might contribute to the development and progression of the inflammatory reactions in COPD patients.

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.

The prevention of TNF-α/IFN-γ mixture-induced inflammation in human keratinocyte and atopic dermatitis-like skin lesions in Nc/Nga mice by mineral-balanced deep sea water

Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by environmental and chemical allergens. Despite the complexity of its pathogenesis, many investigations have shown that substances having anti-inflammatory activities alleviated the pathology of AD. Here, we evaluated the effects of mineral-balanced deep sea water (DSW) on AD-like skin damage in both in vitro and in vivo. The results showed that mineral-balanced DSW regressed inflammatory chemokines, such as macrophage-derived chemokine (MDC), thymus- and activation-regulated chemokine (TARC) and regulated on activation, normal T-cell expressed and secreted (RANTES), and cytokines, interleukin (IL)-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA expression in HaCaT immortal human keratinocyte treated with tumor necrosis factor (TNF)-α/ interferon (IFN)-γ mixture. Furthermore, increased cyclooxygenase (COX)-2 protein expressions were also reversed, filaggrin gene expression was enhanced and decreased involucrin transcriptions was recovered by mineral-balanced DSW in TNF-α/IFN-γ mixture-treated HaCaT human keratinocyte. Moreover, we revealed that the inhibitory effects of mineral-balanced DSW were mediated with the suppression of signal transducer and activator of transcription (STAT) 1 phosphorylation. In animal experiments, we showed that hardness 2000 of mineral-balanced DSW decreased the serum levels of IgE, IL-4, and histamine, and alleviated the severity score and numbers of scratching in dinitrochlorobezene (DNCB)-treated Nc/Nga mice. Furthermore, increased epidermal thickness and mast cell infiltration by DNCB treatment were reversed by the application of hardness 2000 mineral-balanced DSW. Taken together, the present investigation indicates that mineral-balanced DSW is a potent substance with anti-atopic dermatitis activity.

The influence of protein malnutrition on the production of GM-CSF and M-CSF by macrophages

It is well established that protein malnutrition (PM) impairs immune defenses and increases susceptibility to infection. Macrophages are cells that play a central role in innate immunity, constituting one of the first barriers against infections. Macrophages produce several soluble factors, including cytokines and growth factors, important to the immune response. Among those growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). GM-CSF and M-CSF are important to monocyte and macrophage development and stimulation of the immune response process. Knowing the importance of GM-CSF and M-CSF, we sought to investigate the influence of PM on macrophage production of these growth factors. Two-month-old male BALB/c mice were subjected to PM with a low-protein diet (2%) and compared to a control diet (12%) mouse group. Nutritional status, hemogram and the number of peritoneal cells were evaluated. Additionally, peritoneal macrophages were cultured and the production of GM-CSF and M-CSF and mRNA expression were evaluated. To determine if PM altered macrophage production of GM-CSF and M-CSF, they were stimulated with TNF-α. The PM animals had anemia, leukopenia and a reduced number of peritoneal cells. The production of M-CSF was not different between groups; however, cells from PM animals, stimulated with or without TNF-α, presented reduced capability to produce GM-CSF. These data imply that PM interferes with the production of GM-CSF, and consequently would affect the production and maturation of hematopoietic cells and the immune response.

Interleukin-33 stimulates GM-CSF and M-CSF production by human endothelial cells.

Interleukin (IL)-33, a member of the IL-1 family of cytokines, is involved in various inflammatory conditions targeting amongst other cells the endothelium. Besides regulating the maturation and functions of myeloid cells, granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) have been shown to play a role in such pathologies too. It was the aim of our study to investigate a possible influence of IL-33 on GM-CSF and M-CSF production by human endothelial cells. IL-33, but not IL-18 or IL-37, stimulated GM-CSF and M-CSF mRNA expression and protein production by human umbilical vein endothelial cells (HUVECs) and human coronary artery ECs (HCAECs) through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in an IL-1-independent way. This effect was inhibited by the soluble form of ST2 (sST2), which is known to act as a decoy receptor for IL-33. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor fluvastatin could also be shown to moderately reduce the IL-33-mediated effect on M-CSF, but not on GM-CSF expression. In addition, IL-33, IL-1β, GM-CSF and M-CSF were detected in endothelial cells of human carotid atherosclerotic plaques using immunofluorescence. Upregulation of GM-CSF and M-CSF production by human endothelial cells, an effect that appears to be mediated by NF-κB and to be independent of IL-1, may be an additional mechanism through which IL-33 contributes to inflammatory activation of the vessel wall.

The Toll-like receptor 4-activated neuroprotective microglia subpopulation survives via granulocyte macrophage colony-stimulating factor and JAK2/STAT5 signaling

Toll-like receptor (TLR) 4 mediates inflammation and is also known to trigger apoptosis in microglia. Our time-lapse observations showed that lipopolysaccharide (LPS) stimulation induced rapid death in primary cultures of rat microglia, while a portion of the microglia escaped from death and survived for much longer than 2 days, in which time, all of the control cells had died. However, it remains unclear how the LPS-stimulated microglia subpopulation could continue to survive in the absence of any supplied growth factors. In the present study, to clarify the mechanism underlying the LPS-stimulated survival, we investigated whether microglia could produce their own survival factors in response to LPS, focusing on macrophage colony-stimulating factor (M-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-34, which are mainly supplied by astrocytes or neurons. The LPS-stimulated microglia drastically induced the expression of the GM-CSF mRNA and protein, while M-CSF and IL-34 levels were unchanged. The surviving microglia also significantly upregulated the expression of GM-CSF receptor (GM-CSFR) mRNA without affecting M-CSFR. As for the GM-CSFR downstream signal, LPS resulted in the phosphorylation of STAT5 and its translocation to the nucleus in the surviving microglia. Moreover, a specific JAK2 inhibitor, NVP-BSK805, suppressed STAT5 phosphorylation and microglia survival in response to LPS, indicating a critical role of the JAK2/STAT5 pathway in this survival mechanism. Together, these results suggest that a subpopulation of TLR4-activated microglia may survive by producing GM-CSF and up-regulating GM-CSFR. This autocrine GM-CSF pathway may activate the JAK2/STAT5 signaling pathway, which controls the transcription of survival-related genes. Finally, these surviving microglia may have neuroprotective functions because the neurons remained viable in co-cultures with these microglia.

The effect of dimethyl fumarate on gene expression and the level of cytokines related to different T helper cell subsets in peripheral blood mononuclear cells of patients with psoriasis.

Fumaric acid esters such as dimethyl fumarate (DMF) have proven to be effective in the treatment of psoriasis. In view of the role of Th17 in the pathogenesis of psoriasis, the present study was conducted to investigate the effects of DMF on Th1, Th2, and Th17 responses in patients. Peripheral blood mononuclear cells (PBMCs) were isolated from psoriasis patients and healthy individuals and were cultured in the presence or absence of phytohemagglutinin and DMF. The cell supernatants were removed to measure cytokine secretion, and the lymphocytes were used for real-time polymerase chain reaction to establish gene expression. An increase in gene expression of interferon-γ (IFN-γ), as a marker for Th1 activity, and interleukin-17 (IL-17), granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-22 representing the Th17 subset in the PBMCs of patients in comparison with those of control subjects was observed. Culture of PBMCs from psoriasis patients and controls in the presence of DMF decreased IFN-γ and increased IL-4 gene expression in both groups. Treatment with DMF could significantly decrease IL-17, GM-CSF, and IL-22 mRNA levels in the PBMCs of patients. Decreased release of IFN-γ and GM-CSF cytokine secretion after DMF treatment was also observed in PBMC cultures of patients and controls. These data show the effectiveness of DMF in modulating Th17 cells in addition to Th1/Th2 cells and reflect one of the underlying mechanisms of action of DMF in psoriasis. These findings may also support the possible benefits of using fumarate in the treatment of other autoimmune diseases in the pathogeneses of which Th1 and Th17 cells play major roles.

Endothelin receptor B protects granulocyte macrophage colony-stimulating factor mRNA from degradation.

Evidence is lacking on the differential effects of the two therapeutic concepts of endothelin receptor antagonists (ERAs): the blockade of only the endothelin receptor A (ETAR; selective antagonism) versus both ETAR and endothelin receptor B (ETBR; dual blockade). Ambrisentan, a selective ERA, and bosentan, a dual blocker, are both available for therapy. We hypothesized that there are differences in the potential of ERAs to ameliorate inflammatory processes in human airway smooth muscle cells (HASMCs) and aimed to unravel underlying mechanisms. We used HASMC culture, enzyme-linked immunosorbent assay, and quantitative reverse-transcription polymerase chain reaction. Tumor necrosis factor α (TNFα) induced transcription and expression of chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), granulocyte macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinase 12 (MMP12) in HASMCs. In concentration-response experiments, bosentan led to a significantly greater reduction of GM-CSF and MMP12 protein release than ambrisentan, whereas there was no significant difference in their effect on GM-CSF and MMP12 mRNA. Both ERAs reduced CXCL3 protein and mRNA equally but had no effect on CXCL2. Blocking mitogen-activated protein kinases revealed that both ETAR and ETBR signal through p38 mitogen-activated protein kinase, but ETBR also signals through extracellular signal-regulated kinase (ERK) 1/2 to induce GM-CSF expression. In the presence of the transcription inhibitor actinomycin D, bosentan, but not ambrisentan, reduced GM-CSF but not MMP12 or CXCL3 mRNA. In conclusion, blockade of each endothelin receptor subtype reduces GM-CSF transcription, but blocking ETBR additionally protects GM-CSF mRNA from degradation via ERK-1/2. Accordingly, blocking both ETAR and ETBR leads to a stronger reduction of TNFα-induced GM-CSF protein expression. This mechanism might be specific to GM-CSF. Our data stress the anti-inflammatory potential of ERA and warrant further investigation of their utility in chronic inflammatory airway diseases.