Granulocyte Colony-stimulating Factor Secretion Research Articles

Subcutaneous G-CSF administration improves IVF outcomes in patients with recurrent implantation failure presenting a KIR/HLA-C mismatch

Research questionDespite advances in assisted reproductive technologies, many blastocysts are lost unexpectedly during implantation. Alterations in maternal immune tolerance towards fetal antigens may contribute to adverse IVF outcomes. The purpose of this study is to evaluate whether administering Granulocyte Colony-Stimulating Factor (G-CSF) to couples with a Human Leukocyte Antigen/Killer-Cell Immunoglobulin-Like Receptor (HLA/KIR) mismatch could positively modulate the implantation process in patients with recurrent implantation failure (RIF). A KIR/HLA-C mismatch occurs when the interaction between KIRs and HLA-C causes an inhibition of NK cells, which may result in reduced G-CSF secretion leading to impaired placentation and increased risk of miscarriage, pre-eclampsia and fetal growth restriction. DesignA retrospective monocentric cohort study conducted at the IVI Clinic in Rome, including women with a history of at least two failed blastocyst transfers. Couples underwent KIR and HLA-C testing. Couples with a KIR/HLA-C mismatch received G-CSF subcutaneously up to week nine of gestation. The mismatch included cases with inhibitory KIR genotypes and HLA-C2C2 females with HLA-C1C1, or C1C2 males or HLA-C1C2 females with male HLA-C2C2. The reproductive outcomes were assessed, and the logistic regression models controlled for potential confounders affecting IVF outcomes. Results79 patients with RIF and a KIR/HLA-C mismatch were included in the study. 30 patients were administered G-CSF, and 49 received no treatment. In the univariate analysis, no statistically significant differences were reported in the reproductive outcomes after IVF between the women treated with G-CSF and the control group. However, the logistic regression analysis that controlled for confounding factors showed that patients treated with subcutaneous G-CSF had statistically significant higher ongoing-pregnancy (aOR=3.808) and live-birth (aOR=4.998) rates, and a lower miscarriage rate (aOR=0.057). No statistically significant differences were found in other reproductive outcomes. ConclusionThe use of subcutaneous G-CSF in patients with a KIR/HLA-C mismatch undergoing IVF may reduce miscarriage and improve live-birth rates. G-CSF may modulate NK-mediated immune mechanisms and improve trophoblast invasion and development. Randomized trials are warranted to validate these findings and enhance the chances of successful pregnancies in couples with an immunological mismatch.

Interactions between Integrin α9β1 and VCAM-1 Promote Neutrophil Hyperactivation and Deep Vein Thrombosis in a Murine Model of Lung Cancer

Background: Venous thromboembolism (VTE) is highly prevalent in patients with lung cancer and is associated with increased morbidity and mortality. Ectopic secretion of granulocyte colony-stimulating factor (G-CSF), a critical regulator of neutrophil trafficking, is commonly observed in lung cancer. In recent years, compelling evidence has emerged implicating neutrophils and neutrophil extracellular traps (NETosis) in cancer-associated thrombosis. Integrin α9β1 is highly expressed on neutrophils, upregulated upon neutrophil activation, promotes NETosis, and is known to stabilize neutrophil adhesion to activated endothelium via multiple ligands including vascular cell adhesion molecule 1 (VCAM-1). Interactions between integrin α9 and VCAM-1 have been reported to reduce neutrophils apoptosis. Given the relevance of integrin α9 in neutrophil migration and apoptosis, granulopoiesis, and thromboinflammation, here we evaluated the mechanistic role of neutrophil integrin α9β1 in the pathophysiology of cancer-associated thrombosis. Methods: Lung cancer was induced by the intravenous administration of 3X10 5 Lewis lung carcinoma cells. Deep vein thrombosis (DVT) was induced in neutrophil-specific α9 -/- (α9 fl/flMrp8Cre +/-) and littermate controls (α9 fl/flMrp8Cre -/-) by partial ligation of the inferior vena cava (IVC). Plasma levels of the thrombin-antithrombin (TAT) complex, fibrinogen, G-CSF, elastase, and myeloperoxidase (MPO) were assessed by ELISA. To evaluate the potential mechanisms by which neutrophil integrin α9-VCAM-1 interactions promote DVT, we performed unbiased RNA sequencing from 1) control neutrophils stimulated with and without VCAM-1 and, 2) neutrophils of α9 fl/flMrp8Cre +/- and α9 fl/flMrp8Cre -/- mice stimulated with VCAM-1. Computational modeling, docking, virtual screening, and the cell-free assay were used to identify small-molecule inhibitors of integrin α9-VCAM-1 interaction. Results: Mice with lung cancer exhibited significantly increased levels of TAT, G-CSF, blood neutrophil/lymphocyte ratio, cell-free DNA, elastase, and MPO, suggesting a hypercoagulable and inflammatory state. Tumor-bearing and non-tumor-bearing neutrophil-specific α9 -/- mice exhibited reduced DVT severity, as evident by reduced IVC thrombus weight and thrombus length compared to littermate controls, while thrombosis incidence was comparable between the groups. Reduced DVT severity in neutrophil-specific α9 -/- mice was also associated with decreased neutrophil content and citrullinated histone H3 (CitH3) expression in IVC thrombi. Integrin α9 deficient neutrophils incubated with VCAM-1 exhibited reduced release of elastase and MPO, decreased endothelial cell apoptosis, and reduced adhesion to the activated endothelium at venous shear rate compared to control neutrophils incubated with VCAM-1. Bioinformatic analysis of RNA sequencing data from VCAM-1 stimulated neutrophils from α9 fl/flMrp8Cre +/- and control mice showed distinct transcriptional profiles. Hierarchical clustering of gene expression revealed gene clusters that were highly downregulated in stimulated neutrophils from α9 fl/flMrp8Cre +/- mice. Gene ontology enrichment revealed that highly downregulated genes corresponded to biological processes involved in the inflammatory response, cytokine production, NF-κB and ERK signaling pathways, granulocyte migration, and chemotaxis. Virtual screening and cell-free assay identified macitentan as a potent inhibitor of integrin α9-VCAM-1 interactions. In vitro treatment with macitentan reduced neutrophil adhesion to VCAM-1 and also to activated endothelial cells at venous shear rate. In vivo, macitentan treatment at a dose of 5 mg/kg exhibited reduced DVT severity, decreased plasma levels of elastase and MPO, and reduced neutrophil and CitH3 levels in IVC thrombi. Conclusion: Using genetic and pharmacological approaches in combination with transcriptomic and pharmacological studies, we described a previously unknown pathway involving integrin α9 and VCAM-1 in promoting cancer-associated thrombosis. We found that integrin α9-VCAM1 axis is critical for neutrophil hyperactivation, and its pharmacological inhibition significantly reduces DVT severity. The results of this study suggest that neutrophil integrin α9 is a key factor regulating neutrophil hyperactivation and cancer-associated thrombosis.

Macrophage polarization in placenta accreta and macrophage-trophoblast interactions.

Placenta accreta (PA) is defined by an abnormal invasion of placental trophoblasts into the myometrium, which can lead to serious postpartum complications. Macrophages play an important role in the regulation of trophoblast function. Both granulocyte colony-stimulating factor (G-CSF) and its receptor (granulocyte colony-stimulating factor receptor, G-CSFR) have effects on trophoblast invasion. However, the current understanding of G-CSF secretion, G-CSFR expression, abnormal polarization of decidual macrophages (dMϕ) in PA and the abnormal invasion of placental trophoblasts into the myometrium are limited. The polarization of dMϕ in PA was analyzed by flow cytometry (FCM), and the expression of G-CSFR in placental trophoblasts in PA was evaluated by immunohistochemistry. In an in vitro co-culture model, we investigated the effects of HTR-8/SVneo trophoblasts cell line (HTR-8) on macrophage human monocyte cell line (THP-1) polarization and G-CSF secretion, and we also analyzed the effects of THP-1 cells, especially M2-like subtype, on primary trophoblasts and HTR-8 proliferation, invasion, and adhesion. FCM, transwell assays, adhesion assays, and proliferation assays were used in the above model. Compared with controls (n = 9), dMϕ showed significantly lower levels of M1 markers CD80 and CD86 and higher levels of the M2 markers CD163 and CD206, and G-CSFR expression of placental trophoblasts was increased in PA (n = 5). In vitro experiments showed that the trophoblast HTR-8 cell line induced polarization of THP-1 cells to an M2-like subtype and increased their secretion of G-CSF. Furthermore, IL-4/IL-13-induced M2-like THP-1 macrophages were able to increase the expression of G-CSFR, proliferation, invasion and adhesion of both primary trophoblasts and HTR-8 trophoblasts. There is an altered immune imbalance at the maternal-fetal interface in PA, which further may lead to abnormal trophoblast function. G-CSF and its receptors may play important roles in abnormal polarization of macrophages and abnormal invasion of trophoblasts.

Association of Hypernatremia with Immune Profiles and Clinical Outcomes in Adult Intensive Care Unit Patients with Sepsis.

Both hypernatremia and an abnormal immune response may increase hospital mortality in patients with sepsis. This study examined the association of hypernatremia with abnormal immune response and mortality in 520 adult patients with sepsis in an intensive care unit (ICU). We compared the mortality and ex vivo lipopolysaccharide (LPS)-induced inflammatory response differences among patients with hyponatremia, eunatremia, and hypernatremia, as well as between patients with acquired hypernatremia on ICU day 3 and those with sustained eunatremia over first three ICU days. Compared with eunatremia or hyponatremia, hypernatremia led to higher 7 day, 14 day, 28 day, and hospital mortality rates (p = 0.030, 0.009, 0.010, and 0.033, respectively). Compared with sustained eunatremia, acquired hypernatremia led to higher 7, 14, and 28 day mortality rates (p = 0.019, 0.042, and 0.028, respectively). The acquired hypernatremia group nonsignificantly trended toward increased hospital mortality (p = 0.056). Day 1 granulocyte colony-stimulating factor (G-CSF) and tumor necrosis factor (TNF) α levels were relatively low in patients with hypernatremia (p = 0.020 and 0.010, respectively) but relatively high in patients with acquired hypernatremia (p = 0.049 and 0.009, respectively). Thus, in ICU-admitted septic patients, hypernatremia on admission and in ICU-acquired hypernatremia were both associated with higher mortality. The higher mortality in patients with hypernatremia on admission was possibly related to the downregulation of G-CSF and TNF-α secretion after endotoxin stimulation. Compared to sustained eunatremia, acquired hypernatremia showed immunoparalysis at first and then hyperinflammation on day 3.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

A NOVEL ORALLY ACTIVE METABOLITE REVERSES CROHN’S DISEASE-ASSOCIATED INTESTINAL FIBROSIS

Abstract BACKGROUND Intestinal fibrosis is a debilitating complication of Crohn’s disease (CD) patients. Our groups and others discovered that intestinal metabolites are associated with intestinal fibrosis. We selected the most relevant metabolites and determined their anti-fibrogenic efficacies in three mouse models of intestinal fibrosis. METHODS We compared the intestinal metabolite profiles between non-IBD, stricturing CD, and non-stricturing CD patients in inflammatory bowel disease (IBD) multi-omics database to identify CD stricture-related metabolites. We compared the fecal metabolite profiles between trinitrobenzene sulfonic acid (TNBS)-treated mice with and without anti-fibrogenic drug CSA13 treatment to identify mouse intestinal fibrosis-related metabolites. RESULTS Compared to non-stricturing CD patients and non-IBD patients, stricturing CD patients had reduced fecal levels of hydrocinnamate (H), inosine (I), taurine (T), tauroursodeoxycholate (TUDCA), and sphingosine (S). Oral CSA13 increased fecal levels of inosine (I) and sphingosine (S) in the TNBS-treated mice. Overall disease activity (ODA) is a comprehensive assessment of histology and fibrosis scores and intestinal mRNA expression of fibrosis and inflammation markers. Sphingosine (10mg/kg/day for 7-14 days via oral gavages) showed the best anti-fibrogenic effects against spontaneous ileal fibrosis in 42-week-old SAMP1/YitFc mice (11% ODA), cecal fibrosis in Salmonella-infected mice (10% ODA), and colonic fibrosis in TNBS-treated mice (9% ODA). The other four metabolites were ineffective in reversing intestinal fibrosis. Sphingosine (10 microM), but not other metabolites, directly inhibited collagen expression in human colonic CCD-18Co fibroblasts and primary human intestinal fibroblasts from CD patients (CD-HIF). RNA-seq and proteomics identified that low MAPK3 and high ZEB1 mRNA expression in intestines are associated with intestinal fibrosis in CD patients. Sphingosine activated ERK1/2 phosphorylation and inhibited TGF-b1-induced ZEB1 expression in CCD-18Co fibroblasts. The sphingosine-mediated inhibition of collagen synthesis was reversed by ERK1/2 inhibitor PD98059 and ZEB1 overexpression. The anti-fibrogenic effect of sphingosine in the TNBS model was reversed by Zeb1 overexpression. Stricturing CD patients have higher circulating granulocyte-colony stimulating factor (G-CSF) levels than non-stricturing CD patients. Csf3 promoted collagen expression in CD-HIF, while Csf3 inhibition diminished cecal fibrosis in Salmonella-infected mice. Sphingosine diminished G-CSF secretion in primary human peripheral blood mononuclear cells (PBMCs). Sphingosine also inhibited cecal Csf3 mRNA expression and reduced circulating G-CSF levels in Salmonella-infected mice. CONCLUSIONS Sphingosine exerts the most potent anti-fibrogenic effects by modulating ERK1/2, ZEB1, and G-CSF expression.

Effect of steam-processing of the Panax ginseng root on its inducible activity on granulocyte-colony stimulating factor secretion in intestinal epithelial cells in vitro

Ethnopharmacological relevancePanax ginseng root has been used as tonic in traditional Chinese medicine (TCM) and traditional Japanese Kampo medicine. Steam processing of Panax ginseng root is carried out to enhance its nourishing effects on qi.Aim of the study: In order to explore the mechanism of these beneficial effects behind the steam processing of the P. ginseng root, we evaluated effectiveness of processing on the granulocyte-colony stimulating factor (G-CSF) secretion in intestinal epithelial cell-like MCE301 cells. Materials and methodsWe collected P. ginseng root samples in the markets of China and Japan. Fresh or dried samples were steamed for different time lengths and subsequently dried and extracted. MCE301 cells were incubated with the medium containing various P. ginseng root extracts, while the concentration of G-CSF in the medium was measured. We also investigated the active ingredients by size exclusion HPLC. ResultsThe extracts of fresh P. ginseng hairy root samples steamed for more than 6 h significantly induced G-CSF secretion, and the maximum activity was recorded at a 9-h steaming. The same activity was noted when already dried P. ginseng hairy root samples were steamed. The extracts of fresh P. ginseng hairy root without steam processing and those of fresh P. ginseng root body samples with steam processing exhibited no activities. The active ingredients of steamed P. ginseng hairy root samples were high-molecular-weight compounds with an average molecular weight of 758 kDa, and the activity was mediated by the toll-like receptor (TLR) 9. ConclusionsOur results shed on more light on the mechanism underlying the appearance of immunostimulatory activity of the P. ginseng hairy root induced by steam processing.

Curcumin inhibits the growth of liver cancer by impairing myeloid-derived suppressor cells in murine tumor tissues.

Curcumin, one of the active ingredients of Curcuma longa (Jianghuang), has been reported to exert multiple bioactivities, including pro-apoptotic and anti-inflammatory activities. In recent years, curcumin has been extensively studied, and it has been revealed that curcumin inhibits the growth of numerous types of cancer. However, to the best of our knowledge, the inhibitory effects of curcumin on the activation or expansion of myeloid-derived suppressor cells (MDSCs) in liver cancer and the underlying mechanism have not yet been determined. Therefore, the present study aimed to investigate the inhibitory effect of curcumin on MDSC activity and the associated anti-neoplastic mechanism in a HepG2 ×enograft mouse model. The effect of curcumin on the viability of Huh-7, MHCC-97H and HepG2 cells in vitro was analyzed using a Cell Counting Kit-8 assay. The effects of curcumin on tumor growth, numbers of MDSCs, expression levels of proteins involved in the toll-like receptor 4 (TLR4)/NF-κB signaling pathway, levels of related inflammatory factors and angiogenesis were determined in HepG2 ×enograft model mice, which were given different doses of curcumin via intragastrical administration. The results of the present study revealed that curcumin inhibited the viability of Huh-7, MHCC-97H and HepG2 cells and the growth of HepG2 ×enograft tumors in mice. Flow cytometric analysis indicated that curcumin reduced the number of MDSCs in mouse xenograft tumors. In addition, the results demonstrated that curcumin inhibited the TLR4/NF-κB signaling pathway and the expression of inflammatory factors, including IL-6, IL-1β, prostaglandin E2 and cyclooxygenase-2, in mouse xenograft tumors. Furthermore, curcumin suppressed the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF), which are essential factors for MDSCs modulation, in tumor tissues. Additionally, curcumin was revealed to inhibit angiogenesis, which was demonstrated by the downregulation of the expression levels of vascular endothelial growth factor, CD31 and α-smooth muscle actin in western blotting, immunohistochemistry and immunofluorescence experiments. In conclusion, the findings of the present study identified a novel mechanism via which curcumin may suppress the growth of liver cancer by reducing the numbers of MDSCs and subsequently disrupting the process of angiogenesis. These conclusions were supported by the observed inactivation of the TLR4/NF-κB signaling pathway-mediated inflammatory response and the downregulation of GM-CSF and G-CSF secretion in xenograft tissues.

Vitamin D Promotes Trophoblast Cell Induced Separation of Vascular Smooth Muscle Cells in Vascular Remodeling via Induction of G-CSF.

Vitamin D deficiency is associated with complications of pregnancy such as pre-eclampsia, fetal growth restriction, and miscarriage, all of which are also associated with incomplete spiral artery (SpA) remodeling. We have previously shown that both uterine natural killer (uNK) cells and extravillous trophoblast cells (EVT) are required for successful SpA remodeling, but whether their activity in this process is modulated by vitamin D is not known. In the current study, we use a previously described chorionic plate artery (CPA) ex vivo model of vascular remodeling to determine the effects of 1,25(OH)2D treated uNK cell, placental explant (PEx), and uNK/PEx conditioned medium (CM) on vascular smooth muscle cell (VSMC) disorganization and phenotypic switching. Significant results were followed up in VSMCs in vitro. We demonstrate that 1,25(OH)2D can enhance the ability of PEx to induce SpA remodeling, via a mechanism associated with increased secretion of granulocyte-colony stimulating factor (G-CSF). G-CSF appears able to increase VSMC disorganization and phenotypic switching in both an ex vivo vascular model and in vitro VSMC cultures. The clinical relevance of these findings are still to be determined. G-CSF may have differential effects depending on dose and vascular bed, and vitamin D may play a role in potentiating these actions. G-CSF may be an interesting potential therapeutic target for facilitating physiological vascular remodeling for the prevention of adverse obstetric outcomes.

Differential role of segments of \u03b1-mating factor secretion signal in Pichia pastoris towards granulocyte colony-stimulating factor emerging from a wild type or codon optimized copy of the gene

BackgroundThe methylotrophic yeast, Pichia pastoris has been widely used for the production of human therapeutics, but production of granulocyte colony-stimulating factor (G-CSF) in this yeast is low.The work reported here aimed to improve the extracellular production of G-CSF by introducing mutations in the leader sequence and using a codon optimized copy of G-CSF. Bioinformatic analysis was carried out to propose an explanation for observed effect of mutations on extracellular G-CSF production.ResultsMutations in the pro-region of the α-mating type (MAT) secretory signal, when placed next to a codon optimized (CO)-GCSF copy, specifically, the Δ57–70 type, led to highest G-CSF titre of 39.4 ± 1.4 mg/L. The enhanced effect of this deletion was also observed when it preceded the WT copy of the gene. Deletion of the 30–43 amino acids in the pro-peptide, fused with the wild type (WT)-GCSF copy, completely diminished G-CSF secretion, while no effect was observed when this deletion was in front of the CO-GCSF construct. Also, Matα:Δ47–49 deletion preceding the WT-GCSF dampened the secretion of this protein, while no effect was seen when this deletion preceded the CO-GCSF copy of the gene. This indicated that faster rates of translation (as achieved through codon optimization) could overcome the control exercised by these segments. The loss of secretion occurring due to Δ30–43 in the WT-GCSF was partially restored (by 60%) when the Δ57–70 was added. The effect of Δ47–49 segment in the WT-GCSF could also be partially restored (by 60%) by addition of Δ57–70 indicating the importance of the 47–49 region. A stimulatory effect of Δ57–70 was confirmed in the double deletion (Matα:Δ57–70;47–49) construct preceding the CO-GCSF. Secondary and tertiary structures, when predicted using I-TASSER, allowed to understand the relationship between structural changes and their impact on G-CSF secretion. The Δ57–70 amino acids form a major part of 3rd alpha-helix in the pre-pro peptide and its distortion increased the flexibility of the loop, thereby promoting its interaction with the cargo protein. A minimum loop length was found to be necessary for secretion. The strict control in the process of secretion appeared to be overcome by changing the secondary structures in the signal peptides. Such fine tuning can allow enhanced secretion of other therapeutics in this expression system.ConclusionsAmong the different truncations (Matα:Δ57–70, Matα:Δ47–49, Matα:Δ30–43, Matα:Δ57–70;30–43, Matα:Δ57–70;47–49) in pro-peptide of α-MAT secretion signal, Matα:Δ57–70 fused to CO-GCSF, led to highest G-CSF titre as compared to other Matα truncations. On the other hand, Matα:Δ30–43 and Matα:Δ47–49 fused to the WT-GCSF dampened the secretion of this protein indicating important role of these segments in the secretion of the cargo protein.

Honey isomaltose contributes to the induction of granulocyte-colony stimulating factor (G-CSF) secretion in the intestinal epithelial cells following honey heating

We have previously discovered that heated honey but not unheated honey could induce the secretion of granulocyte-colony stimulating factor (G-CSF) in the MCE301 intestinal epithelial cells. The objective of this study was to identify compounds in honey that could contribute to this activity. We bought several kinds of commercial honey samples derived from different flowers, as well as corn syrup samples, in the markets of China and Japan, and heated them at 180 °C for 30 min. MCE301 cells were treated with the medium containing the samples, and G-CSF levels in the medium were measured by ELISA. By comparing their activities and sugar contents, we discovered that isomaltose was primarily implicated. The optimum heating conditions for isomaltose were at 180 °C for 60 min or at 200 °C for 15–30 min, and these time- and temperature-dependencies were similar to those of honey in our previous study. When heated isomaltose was partitioned by dialysis, the active ingredients were transferred into a high-molecular-weight fraction. By size-exclusion HPLC analysis, the average molecular weight of heated isomaltose was 790 kDa. When heated isomaltose was hydrolyzed by acids, glucose was subsequently produced. Maltose, sucrose, turanose, and trehalose did not exhibited any activity when heated at 180 °C for 60 min, indicating that the glucose groups with α(1 → 6)-binding in the isomaltose molecule play important roles in its activity when oxidatively polymerized by heat. The stimulating activity of heated isomaltose was inhibited by toll-like receptor 4 (TLR4) inhibitor, suggesting that heated isomaltose activates TLR4 to induce G-CSF. Since G-CSF is clinically used for cancer patients to accelerate their recovery from neutropenia following chemotherapy or accompanied with aplastic anemia, these findings indicate that honey which contains high level of isomaltose could improve immunosuppressive conditions when honey is heated, and that heated isomaltose might be of potential therapeutic use in patients with compromised immunity caused by chemotherapeutic agents.

HLA-G LEVELS IN FOLLICULAR FLUID ARE NOT ASSOCIATED WITH FOLLICULAR G-CSF CONCENTRATIONS IN WOMEN UNDERGOING IN VITRO FERTILIZATION

Recently, soluble human leukocyte antigen-G (sHLA-G) and granulocyte colony-stimulating factor (G-CSF) quantified in the follicular fluid have been proposed as non-invasive biomarkers of oocyte competence in both stimulated and natural IVF/ICSI cycles. sHLA-G, G-CSF and their receptors were described in various human fetal tissues and it was suggested that they play an important role in ovulation, oocyte maturation, embryo development and implantation. However, the possible association between these two molecules in the follicular fluid has not been studied yet. The purpose of the present study was to determine whether the sHLA-G levels are associated with the G-CSF levels in follicular fluid of women undergoing in vitro fertilization. This is a cohort study of 56 women undergoing stimulated cycles and in vitro fertilization between September 2019 and November 2019 at Nadezhda Women’s Health Hospital. Follicular fluid from up to six clear follicular aspirates was collected during regular egg collection procedure of 56 patients. Samples were stored at -20°C until measurement. Follicular fluid levels of sHLA-G and G-CSF were evaluated by ELISA kits (Elabscience; E-EL-H1663 and RayBiotech; ELH-GCSF-1, respectively) according to the manufacturer’s instructions. The absorption was measured on a microplate reader (Beckman Coulter DTX 880 Multimode detector) at 450 nm. Statistical analysis was performed using SPSS v.21 (IBM Corp., Armonk, NY, USA). sHLA-G was detected in all 56 follicular fluid samples, with a median value of 47.52 IU/ml and a range of 13.85-63.06 IU/ml. Follicular fluid G-CSF was also detected in all samples and ranged between 3 and 116 pg/ml with a median of 16.23 pg/ml. Measured sHLA-G levels did not show a significant correlation with G-CSF concentration in follicular fluid (P = 0.63). This is the first report regarding the association between follicular fluid levels of sHLA-G and G-CSF. Although the increased secretion of both sHLA-G and G-CSF usually correlates with better oocyte quality and implantation success, our findings suggest that they are not related to each other.

A paracrine interaction between granulosa cells and leukocytes in the preovulatory follicle causes the increase in follicular G-CSF levels

ObjectiveFollicular granulocyte colony-stimulating factor (G-CSF) is a new biomarker of oocyte quality and embryo implantation in in vitro fertilization (IVF) cycles. Its role in reproduction is poorly understood. Our study aimed to investigate the mechanisms and cells responsible for G-CSF production in the preovulatory follicle.DesignLaboratory research study.SettingSingle-center study.InterventionsGranulosa cells and leukocytes were isolated from the follicular fluids (FF) or the blood of women undergoing IVF and from the blood of a control group of women with spontaneous ovulatory cycles to perform cocultures.Main outcome measureG-CSF-secreted protein was quantified in the conditioned media of cocultures.ResultsG-CSF secretion was considerably increased in cocultures of granulosa cells and leukocytes. This effect was maximal when leukocytes were isolated from the blood of women in the late follicular phase of the menstrual cycle or from the FF of women undergoing IVF. The leukocyte population isolated from the FF samples of women undergoing IVF had a higher proportion of granulocytes than that isolated from the corresponding blood samples. Leukocytes induced the synthesis and secretion of G-CSF by granulosa cells. Among a range of other FF cytokines/chemokines, only growth-regulated oncogene alpha (GROα) was also increased.ConclusionThe notable rise in G-CSF at the time of ovulation coincides with the accumulation of follicular granulocytes, which stimulate G-CSF production by granulosa cells via paracrine interactions. High follicular G-CSF concentrations may occur in follicles with optimal granulosa–leukocyte interactions, which could explain the increased implantation rate of embryos arising from these follicles.

Historical and pharmacological studies on rehmannia root processing– Trends in usage and comparison of the immunostimulatory effects of its products with or without steam processing and pretreatment with liquor

Ethnopharmacological relevanceThe dried root of Rehmannia glutinosa (RR) is a crude drug used in traditional Japanese Kampo medicine and traditional Chinese medicine (TCM). Sometimes, the crude drug is subjected to additional processing before use. Aim of the studyTo determine the effects of steam processing and pretreatment with liquor of RR through historical investigation, analytical chemistry, and pharmacological experiments. Materials and methodsWe inspected TCM literature from the Later Han Dynasty to the present day. Dried RR steamed for 3, 6, 9, or 12 h (steamed RRs, SRRs), dried RR soaked in yellow rice wine (liquor) (liquor-RR), and dried RR steamed for 6 h pretreated with liquor (liquor-SRR) were prepared. These samples were extracted using boiling water, and the inducible effects of the extracts on granulocyte colony-stimulating factor (G-CSF) secretion in cultured enterocytes and the content of their marker compounds were evaluated by using HPLC. ResultsThe effect of processing using both steaming and the pretreatment using liquor described in TCM literature over different eras was to enhance the warming effect and tonifying qi (energy) of RR. We found that SRR, processed by pretreatment with liquor, became mainstream since the Qing Dynasty. In SRR, stachyose content was decreased and fructose and manninotriose contents were increased with steaming time. However, the content of these compounds was not altered by pretreatment with liquor. RR extract induced G-CSF secretion in cultured enterocytes; moreover, the SRR extract steamed for more than 6 h had significantly stronger effects than that RR. Pretreatment with liquor did not cause any significant differences in the effects of RR or SRR. ConclusionsThe aim of processing for RR by both steaming and pretreatment with liquor in TCM literature over different eras was to enhance the tonifying effect on qi and its immunostimulatory effect. Although the effect of RR on the induction of G-CSF secretion in intestinal epithelial cells was enhanced by steaming, this enhancement was not enhanced by the pretreatment with liquor. These results provide scientific support for steaming, but could not elucidate a reason for pretreatment with liquor in TCM theory.

Protease-activated receptor 2 (PAR2) upregulates granulocyte colony stimulating factor (G-CSF) expression in breast cancer cells

Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor which is activated upon cleavage of its N-terminal region. PAR2 has been associated with many aspects regarding tumor progression, such as the production of pro-tumoral cytokines. Granulocyte colony-stimulating factor (G-CSF) is a cytokine essential to neutrophil production and maturation, and it is often overexpressed in tumors. In this study, we evaluated the ability of PAR2 to modulate G-CSF expression. PAR2 and G-CSF were significantly more expressed in metastatic (4T1 and MDA-MB-231) as compared to non-metastatic (67NR and MCF7) breast cancer cell lines. In addition, PAR2 stimulation by a synthetic agonist peptide significantly increased G-CSF gene expression in the metastatic cell lines. Knockdown of PAR2 in 4T1 cells decreased G-CSF expression and secretion. In addition, treatment of 4T1 with the commercial PAR2 antagonist, ENMD-1068, significantly decreased G-CSF expression. cBioPortal analyses of the TCGA database showed a significant co-occurrence of G-CSF and PAR2 gene overexpression in breast cancer samples. In conclusion, our data suggest that PAR2 contributes to G-CSF expression in breast cancer cells, possibly favoring tumor progression.

Comparison of the inducible effects of licorice products with or without heat-processing and pre-treatment with honey on granulocyte colony-stimulating factor secretion in cultured enterocytes

Ethnopharmacological relevanceLicorice (the roots and rhizomes of Glycyrrhiza uralensis Fisch.) is occasionally used as crude drug following processing including roasting or honey-roasting (soaking with honey before roasting) in traditional Japanese Kampo medicine and traditional Chinese medicine (TCM). Aim of the studyWe investigated the differences in the inducible effect of processed licorice products on granulocyte colony-stimulating factor (G-CSF) secretion in cultured intestinal epithelial cells and elucidated the active ingredients in both unprocessed and processed licorice products. Materials and methodsWe prepared heat-processed licorice with or without pretreatment with honey, and fractionated the extracts by Sephadex G-100. Enterocyte-like differentiated MCE301 cells were incubated in media comprising a hot water extract of licorice products for 24h, and the concentrations of G-CSF in the media were measured using enzyme-linked immunosorbent assay (ELISA). ResultsLicorice extract induced G-CSF secretion in MCE301 cells, and the active ingredients of licorice were high molecular compounds. Although the roasted licorice extract exhibited the activity similar to that of the unprocessed licorice extract, honey-roasted licorice extracts exhibited a significantly higher inducible effect on G-CSF secretion in the cells than that of unprocessed or roasted licorice extracts without pretreatment with honey. This enhanced activity was dependent on the temperature and heating time. ConclusionsThe enhanced inducible effect of honey-roasted licorice on G-CSF secretion might be attributed to the combined effect of licorice-derived high molecular compounds and heated-honey-derived compounds. The results of this study can scientifically explain the objective of processing via honey-roasting in TCM theory.

Hyperglycemia Impairs Neutrophil Mobilization Leading to Enhanced Metastatic Seeding

Preexisting diabetes is a risk factor for the development of multiple types of cancer. Additionally, diabetic patients face a poorer prognosis when diagnosed with cancer. To gain insight into the effects of hyperglycemia, a hallmark of diabetes, on tumor growth and metastatic progression, we combined mouse models of cancer and hyperglycemia. We show that while hyperglycemia attenuates primary tumor growth, it concomitantly increases metastatic seeding in a distant organ. We further show that the increase in metastatic seeding is due to impaired secretion of granulocyte colony-stimulating factor (G-CSF) and impaired neutrophil mobilization. Normalizing blood glucose levels using insulin rescues neutrophil recruitment and tumor growth and concomitantly reduces metastatic seeding. These results provide links among hyperglycemia-induced changes in neutrophil mobilization, primary tumor growth, and metastatic progression. Furthermore, our observations highlight the importance of normalizing blood glucose levels in hyperglycemic cancer patients.

Comparison of byakujutsu (Atractylodes rhizome) and sojutsu (Atractylodes lancea rhizome) on anti-inflammatory and immunostimulative effects in vitro.

The Japanese Pharmacopoeia defines byakujutsu (Atractylodes rhizome) as the rhizome of Atractylodes japonica or A. macrocephala and sojutsu (Atractylodes lancea rhizome) as the rhizome of A. lancea, A. chinensis, or their interspecific hybrids. Because their pharmaceutical uses differ in traditional Japanese Kampo medicine and traditional Chinese medicine, with less apparent scientific evidence, we compared the pharmacological properties between byakujutsu and sojutsu. Crude drug specimens of byakujutsu (n=40) and sojutsu (n=49) obtained in markets were identified by their species using DNA profiling. Their pharmacological properties were evaluated by the inhibitory effect of a MeOH extract of the samples on nitric oxide (NO) production by lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells and by the inducing effect of boiling water extract of the samples on granulocyte-colony stimulating factor (G-CSF) secretion from murine normal colonic epithelial MCE301 cells. We authenticated A. macrocephala (n=8), A. japonica (n=35), and the hybrid between A. macrocephala and A. japonica (n=1), and they were used as byakujutsu. We authenticated A. chinensis (n=25), A. lancea (n=14), and the hybrid between A. chinensis and A. lancea (n=6), and they were used as sojutsu. The inhibitory effects of byakujutsu on NO production were significantly higher than those of sojutsu (P<0.05). This activity of A. japonica rhizome was significantly higher than that of A. macrocephala rhizome and A. lancea rhizome (P<0.01). The activity of A. chinensis rhizome was significantly higher than that of A. lancea rhizome (P<0.05). The extract of A. japonica rhizome significantly induced G-CSF secretion from MCE301 cells in a concentration-dependent manner. These effects of byakujutsu samples were not significantly different from those of sojutsu samples. A. japonica rhizome had significantly higher activity than A. macrocephala rhizome; however, there were no statistically significant differences among A. japonica, A. chinensis, and A. lancea. The pharmacological differences of byakujutsu and sojutsu may not be large among highly variated crude drug samples with average values, and quality control with the identification of the original plant species of byakujutsu and sojutsu may guarantee their pharmacological properties.

Development of Aggressive Pancreatic Ductal Adenocarcinomas Depends on Granulocyte Colony Stimulating Factor Secretion in Carcinoma Cells.

The survival rate for pancreatic ductal adenocarcinoma (PDAC) remains low. More therapeutic options to treat this disease are needed, for the current standard of care is ineffective. Using an animal model of aggressive PDAC (Kras/p48TGFβRIIKO), we discovered an effect of TGFβ signaling in regulation of G-CSF secretion in pancreatic epithelium. Elevated concentrations of G-CSF in PDAC promoted differentiation of Ly6G+ cells from progenitors, stimulated IL10 secretion from myeloid cells, and decreased T-cell proliferation via upregulation of Arg, iNOS, VEGF, IL6, and IL1b from CD11b+ cells. Deletion of csf3 in PDAC cells or use of a G-CSF-blocking antibody decreased tumor growth. Anti-G-CSF treatment in combination with the DNA synthesis inhibitor gemcitabine reduced tumor size, increased the number of infiltrating T cells, and decreased the number of Ly6G+ cells more effectively than gemcitabine alone. Human analysis of human datasets from The Cancer Genome Atlas and tissue microarrays correlated with observations from our mouse model experiments, especially in patients with grade 1, stage II disease. We propose that in aggressive PDAC, elevated G-CSF contributes to tumor progression through promoting increases in infiltration of neutrophil-like cells with high immunosuppressive activity. Such a mechanism provides an avenue for a neoadjuvant therapeutic approach for this devastating disease. Cancer Immunol Res; 5(9); 718-29. ©2017 AACR.

Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro

BackgroundInflammation is a key contributor to central nervous system (CNS) injury such as stroke, and is a major target for therapeutic intervention. Effective treatments for CNS injuries are limited and applicable to only a minority of patients. Stem cell-based therapies are increasingly considered for the treatment of CNS disease, because they can be used as in-situ regulators of inflammation, and improve tissue repair and recovery. One promising option is the use of bone marrow-derived mesenchymal stem cells (MSCs), which can secrete anti-inflammatory and trophic factors, can migrate towards inflamed and injured sites or can be implanted locally. Here we tested the hypothesis that pre-treatment with inflammatory cytokines can prime MSCs towards an anti-inflammatory and pro-trophic phenotype in vitro.MethodsHuman MSCs from three different donors were cultured in vitro and treated with inflammatory mediators as follows: interleukin (IL)-1α, IL-1β, tumour necrosis factor alpha (TNF-α) or interferon-γ. After 24 h of treatment, cell supernatants were analysed by ELISA for expression of granulocyte-colony stimulating factor (G-CSF), IL-10, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), IL-1 receptor antagonist (IL-1Ra) and vascular endothelial growth factor (VEGF). To confirm the anti-inflammatory potential of MSCs, immortalised mouse microglial BV2 cells were treated with bacterial lipopolysaccharide (LPS) and exposed to conditioned media (CM) of naïve or IL-1-primed MSCs, and levels of secreted microglial-derived inflammatory mediators including TNF-α, IL-10, G-CSF and IL-6 were measured by ELISA.ResultsUnstimulated MSCs constitutively expressed anti-inflammatory cytokines and trophic factors (IL-10, VEGF, BDNF, G-CSF, NGF and IL-1Ra). MSCs primed with IL-1α or IL-1β showed increased secretion of G-CSF, which was blocked by IL-1Ra. Furthermore, LPS-treated BV2 cells secreted less inflammatory and apoptotic markers, and showed increased secretion of the anti-inflammatory IL-10 in response to treatment with CM of IL-1-primed MSCs compared with CM of unprimed MSCs.ConclusionsOur results demonstrate that priming MSCs with IL-1 increases expression of trophic factor G-CSF through an IL-1 receptor type 1 (IL-1R1) mechanism, and induces a reduction in the secretion of inflammatory mediators in LPS-activated microglial cells. The results therefore support the potential use of preconditioning treatments of stem cells in future therapies.