Macrophage Colony Research Articles

Addition of granulocyte macrophage colony stimulating factor (GM-CSF) during in vitro oocyte maturation improves embryo development in a mouse model of advanced maternal age.

Oocyte developmental competence declines in women aged 35 and older resulting in many women resorting to IVF. The present study determined whether adding Granulocyte-macrophage colony-stimulating factor (GM-CSF) during in vitro oocyte maturation (IVM) could improve oocyte developmental competence in a mouse model of advanced maternal age. Oocytes from 12-14 month C57BL6 J × CBA mice were treated with 10 ng/ml of GM-CSF during IVM, and embryo development, mitochondrial activity, spindle formation and chromosomal alignment were examined. The addition of GM-CSF tended to increase fertilisation rates (76.19 vs. 82.03%; P = 0.07) but did not affect cumulus expansion compared with control. The addition of GM-CSF also increased blastocysts rates (51.10 vs. 61.52%; P < 0.01) and the number of good quality blastocysts (33.31 vs. 44.13%; P < 0.05) present at 96 h of culture as well as inner cell mass (12.64 vs. 15.62; P < 0.01) and total cell number (42.98 vs. 48.78; P < 0.05). GM-CSF treatment also increased mitochondrial membrane potential two to three fold in the outer (2.86 vs. 0.97; P < 0.001), intermediate (3.25 vs. 0.89; P < 0.001) and peri nuclear areas (3.62 vs. 1.08; P < 0.001). GM-CSF treatment did not influence spindle formation or chromosomal alignment. Together our results indicate that the addition of GM-CSF during IVM may improve oocyte quality in women of advanced maternal age.

Investigating the influence of titanium particle size and concentration on osteogenic response of human osteoblasts - in vitro study.

The purpose of this study was to investigate the correlation between the size and concentration of titanium particles and the osteogenic response of human osteoblasts (HOB). Different concentrations of titanium dioxide nano- and micro-particles were prepared and their biocompatibility on HOBs was analyzed using XTT assay. The changes in the actin cytoskeletal organization were studied by confocal laser scanning microscopy. The generation of intracellular reactive oxygen species (ROS) by HOBs after exposure to titanium dioxide particles was analyzed using ROS assay. Besides, the osteogenic potential represented by alkaline phosphatase activity, osteoprotegerin, macrophage colony stimulating factor levels, and biomineralization were analyzed. Short-term interaction of titanium dioxide nano- and micro-particles did not induce toxicity to HOBs. However, cells treated with 100 μg/mL titanium dioxide nano- and micro-particles demonstrated higher ROS generation compared to control. Besides, cells treated with 100 μg/mL titanium dioxide nanoparticles showed higher alkaline phosphatase activity, osteoprotegerin, macrophage colony stimulating factor levels and biomineralization compared to titanium dioxide microparticles. Collectively, the study found titanium dioxide nanoparticles to be more biocompatible than microparticles providing an insight into the capability of nanostructures in supporting osteoblast differentiation and its plausibility in biomedical applications.

Granulocyte macrophage colony stimulating factor exerts dominant effects over macrophage colony stimulating factor during macrophage differentiation in vitro to induce an inflammatory phenotype.

Macrophages (Mφ) can exist along a spectrum of phenotypes that include pro-inflammatory (M1) or anti-inflammatory (M2) immune cells. Mφ colony stimulating factor (M-CSF) and granulocyte Mφ colony stimulating factor (GM-CSF) are cytokines important in hematopoiesis, polarization and activation of Mφ. To gain a greater understanding of the relationship between GM-CSF and M-CSF, we investigated an in vitro model of differentiation to determine if GM-CSF and M-CSF can antagonize each other, in terms of Mφ phenotype and functions. We determined that Mφ cultured in mixed M-CSF: GM-CSF ratios exhibit M1-like GM-CSF-treated macrophage phenotype when the ratios of the two cytokines are 1:1 in culture. Moreover, GM-CSF is dominant over M-CSF in influencing Mφ production of proinflammatory cytokines such as IL-6, TNFα, and IL-12p40, and the anti-inflammatory cytokine IL-10. Our data established that GM-CSF is more dominant over M-CSF, triggering the Mφ to become pro-inflammatory cells. These findings provide insight into how GM-CSF can influence Mφ activation with implications in inflammatory diseases where the Mφ status can play a significant role in supporting the inflammatory conditions.

NOTCH2 promotes osteoclast maturation and metabolism and modulates the transcriptome profile during osteoclastogenesis

Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.

Unlocking the potential of dimethyl fumarate: enhancing oncolytic HSV-1 efficacy for wider cancer applications.

Immunotherapy and specifically oncolytic virotherapy has emerged as a promising option for cancer patients, with oncolytic herpes simplex virus-1 (oHSV-1) expressing granulocyte macrophage colony stimulating factor being the first OV to be approved by the FDA for treatment of melanoma. However, not all cancers are sensitive and responsive to oncolytic viruses (OVs). Our group has demonstrated that fumaric and maleic acid esters (FMAEs) are very effective in sensitizing cancer cells to OV infection. Of note, these FMAEs include dimethyl fumarate (DMF, also known as Tecfidera®), an approved treatment for multiple sclerosis and psoriasis. This study aimed to assess the efficacy of DMF in combination with oncolytic HSV-1 in preclinical cancer models. We demonstrate herewith that pre-treatment with DMF or other FMAEs leads to a significant increase in viral growth of oHSV-1 in several cancer cell lines, including melanoma, while decreasing cell viability. Additionally, DMF was able to enhance ex vivo oHSV-1 infection of mouse-derived tumor cores as well as human patient tumor samples but not normal tissue. We further reveal that the increased viral spread and oncolysis of the combination therapy occurs via inhibition of type I IFN production and response. Finally, we demonstrate that DMF in combination with oHSV-1 can improve therapeutic outcomes in aggressive syngeneic murine cancer models. In sum, this study demonstrates the synergistic potential of two approved therapies for clinical evaluation in cancer patients.

A Mendelian randomization study on the causal association of circulating cytokines with colorectal cancer

Background Circulating cytokines have been associated with colorectal cancer (CRC). However, their causal correlation remains undetermined. This investigation uses genetic data to evaluate the mechanism that links circulating cytokines and CRC via Mendelian Randomization (MR). Methods A two-sample MR evaluation was carried out to investigate the mechanism associating circulating cytokines and CRC in individuals of European ancestry. The Genome-wide association studies statistics, which are publically accessible, were used. Eligible instrumental SNPs that were significantly related to the circulating cytokines were selected. Multiple MR analysis approaches were carried out, including Simple Mode, inverse variance weighted (IVW), MR-Egger, Weighted Mode, Weighted Median, and MR pleiotropy residual sum and outlier (MR-PRESSO) methods. Results The evidence supporting the association of genetically predicted circulating levels with the increased risk of CRC was revealed; these included vascular endothelial growth factor (OR = 1.352, 95% CI: 1.019–1.315, P = 0.024), interleukin-12p70 (OR = 1.273, 95% CI: 1.133–1.430, P = 4.68×10−5), interleukin-13 (OR = 1.149, 95% CI: 1.012–1.299, P = 0.028), interleukin-10 (OR = 1.230, 95% CI: 1.013–1.493, P = 0.037), and interleukin-7 (OR = 1.191, 95% CI: 1.023–1.386 P = 0.024). Additionally, MR analysis negative causal association between macrophage colony stimulating factor and CRC (OR = 0.854, 95% CI: 0.764–0.955, P = 0.005). The data from Simple Mode, Weighted Median, MR-Egger, and Weighted Mode analyses were consistent with the IVW estimates. Furthermore, the sensitivity analysis indicated that the presence of no horizontal pleiotropy to bias the causal estimates. Conclusion This investigation identified a causal association between circulating cytokines levels risk of CRC and may provide a deeper understanding of the pathogenesis of CRC, as well as offer promising leads for the development of novel therapeutic targets for CRC.

#211 : The Causal Effects of Circulating Biomarkers of Immunity and Inflammation on Endometriosis: A Bidirectional Mendelian Randomization Analysis

Background and Aims: Endometriosis is a chronic gynecological disease with a high prevalence among reproductive-aged women. Several studies have investigated the causal effect of different serum biomarkers of immunity and inflammation on the occurrence and progression of endometriosis. However, the results of these studies remained inconclusive and even controversial. Herein, this study aims to analyze the causal associations of genetically predicted levels of different circulating biomarkers of immunity and inflammation with the risk of endometriosis using a bidirectional two-sample Mendelian randomization (MR) approach. Methods: The single-nucleotide polymorphisms (SNPs) of exposures which indicate the genetic variants were used as instrumental variables (IVs) and were all obtained from the genome-wide association study (GWAS) of European ancestry from the IEU GWAS database. Besides, we performed bidirectional MR analysis to test the associations. The causal effects were estimated through the inverse-variance weighted method (IVW). The sensitivity examinations of each result after the MR analysis were performed to detect if any heterogeneity and pleiotropy existed. Results: Our results demonstrated that higher interleukin-9 (IL-9) level was associated with the occurrence of endometriosis, while macrophage colony stimulating factor (MCSF) level, TNF-related apoptosis inducing ligand (TRAIL), sE-Selectin and basophil count showed the opposite. Genetically predicted endometriosis was related to higher immunoglobulin E (IgE) level and platelet count. No evidence of bidirectional association was detected on the other serum inflammatory biomarkers with endometriosis. Neither pleiotropy nor heterogeneity was found in our study. Conclusion: Our results suggested that some dysregulated circulating biomarkers of immunity and inflammation is causally associated with the occurrence of endometriosis, and at the same time endometriosis may lead to the abnormality of some of these factors. Our findings provided genetic insights on potential novel strategies in the primary prevention, diagnosis and treatment of endometriosis.

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p&amp;lt;0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p&amp;lt;0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

Role of macrophage colony stimulating factor and interferon regulatory factor 7 in modulating the immune profile of mouse testicular macrophages

Testicular macrophages (TM) are critical for the function of the testis by regulating homeostasis and inflammatory responses. However, the mechanisms by which TM fulfil these roles remain elusive. In this study, we explored the impact of two key testicular microenvironmental factors, namely 25-hydroxycholesterol (25HC), an oxysterol related to sex hormones and macrophage colony-stimulating factor (M-CSF), a factor crucial for macrophage survival and differentiation, on the regulation of the TM phenotype. Specifically, we examined their role in controlling the expression of the transcription factor interferon regulatory factor 7 (Irf7), a factor critical for maintaining the alternative macrophage phenotype. To achieve this, we used an in vitro bone marrow-derived macrophage (BMDM) model as a surrogate for TM to investigate the roles of 25HC and M-CSF in regulating the expression of Irf7 during the polarization of murine TM. M-CSF was identified as the main regulator of Irf7 expression, while 25HC production is a consequence of Irf7 activation in BMDM. In turn, 25HC plays a role in a negative feedback loop on the expression levels of Irf7 in BMDM. Using flow cytometry in Irf7-/- mouse testis the CD64loMHChi TM subpopulation was found to be decreased. Together with lower IL-10 protein levels in Irf7-/- TM this indicates a shift towards an M1-like macrophage profile. In summary, our data indicates that M-CSF could act as an inducer of high Irf7 expression levels in the mouse testis. However, the exact role of the high 25HC concentration in the testis in maintaining the local immune milieu still needs further study.

Decaffeinated coffee and green tea extract inhibit foam cell atherosclerosis by lowering inflammation and improving cholesterol influx/efflux balance through upregulation of PPARγ and miR-155

Background Foam cells are markers of atherosclerosis and characterise advanced atherosclerotic plaque, stimulated by inflammation caused by high lipid levels in macrophages. The combination of decaffeinated coffee and green tea extract (DCGTE) has been suggested to have a role in foam cell inhibition. Objective We investigated the inhibiting role of DCGTE against foam cell formation, through modulation of the inflammation process and cholesterol metabolism in macrophage colony stimulating factor- (M-CSF) and oxidized low-density lipoprotein (oxLDL)-exposed macrophages. Methods Coffee and green tea were extracted by filtration and infusion respectively, and underwent decaffeination using active carbon and blanching methods, respectively. Cells were administered 160/160 and 320/320μg/ml of DCGTE. Foam cell formation was observed using a light microscope after staining with Oil Red O (ORO), and the accumulation of lipids in macrophages with ELISA. Observations of lipid influx and efflux were determined through semiquantitative cluster differentiation 36 (CD36) and ATP binding cassette transporter A1 (ABCA1) expression through immunofluorescence. The inflammation process was quantified using inflammatory/anti-inflammatory markers, e.g., tumor necrosis factor α (TNFα) and interleukin 10 (IL10) with ELISA. Peroxisome proliferator activated response γ (PPARγ) expression and activity were assessed with PCR and ELISA, respectively. The expression of microRNA 155 (miR-155) was examined using qPCR. Results DCGTE at the above concentrations tended to reduce foam cell numbers, significantly inhibited lipid accumulation (p=0.000), reduced CD36 expression (p=0.000) and TNFα secretion (p=0.000) in Raw264.7 exposed to M-CSF 50ng/ml and oxLDL 50μg/ml. PPARγ expression (p=0.00) and activity (p=0.001), miR-155 relative expression (p=0.000), and IL10 production (p=0.000) also increased. Conclusion DCGTE lowered foam cell numbers, possibly through attenuation of the inflammatory process and improvement of lipid/efflux mechanisms in M-CSF and oxLDL-stimulated Raw264.7 cells, via upregulation of PPARγ and miR-155. Our results suggest DCGTE may help prevent atherosclerosis-based diseases.

Abstract 16891: High Molecular Weight Hyaluronan Inhibits Macrophage Phagocytosis

Background: After a myocardial infarction (MI), macrophages are recruited to the site of injury to begin the process of wound healing. Macrophages are important because they clear cellular debris via phagocytosis. This period of acute inflammation in the heart coincides with extracellular matrix (ECM) remodeling. Hyaluronan (HA), one of the most abundant ECM components, accumulates in a high molecular weight form (HA HMW ). The relationship between HA HMW accumulation and macrophage function in is not fully understood. Goal: To determine the extent to which hyaluronan impacts phagocytosis in polarized macrophages. Methods and Results: Tissue sections were collected from mice one-week post-MI and stained for hyaluronan binding protein (HABP) to assess HA accumulation. A significant increase in HA was seen in both male (n=4, p=0.0031) and female (n=4, p=0.0210) MI hearts compared to sham hearts. Separately, bone marrow was isolated from naïve mice and cells were treated in culture with macrophage colony stimulating factor (M-CSF) for 7-10 days. The resultant macrophages were then polarized into a pro-inflammatory (M1) phenotype using LPS and INF-γ, or pro-reparative phenotype (M2) using IL-4 and IL-13. Macrophage polarization was confirmed via qRT-PCR. In additional experiments, macrophages were polarized for 24 hours and then incubated for 30 min with FITC-labeled, IgG-coated latex beads in the presence or absence of 1 μM HA HMW . Flow cytometry was used to assess phagocytosis of the beads. HA HMW treatment resulted in a significant reduction in the percent of FITC positive cells in M1 female (n=6, p=0.005), M2 female (n=5, p=0.0406), and M2 male (n=5, p=0.0314) macrophage subsets indicating a decrease in phagocytosis. No significant changes were seen in the M0 and M1 male macrophage subsets. Additionally, the mean fluorescence intensity (MFI) measured in the FITC channel was significantly decreased in all the female subsets as well as the M2 male macrophages (n=5-6, p&lt;0.05). No significant changes were seen in the M0 and M1 male subsets. Conclusion: HA HMW impairs opsonin-induced phagocytosis in macrophages. Given that HA HMW accumulates post-MI, these findings may partially explain the incomplete immune response in the failing heart.

165 Evaluation of the Use of Prenatal Immune Stimulation on the Ability to Alter Postnatal Immune Function in Weaned Pigs

Abstract One of the most lacking areas where improvements can be made in swine production is in the area of swine health. This study was designed to determine whether exposure to low dose endotoxin (lipopolysaccharide; LPS) during gestation can enhance immunity to a subsequent LPS challenge in piglets after weaning. Pregnant Camborough sows (parity: 2.6 ± 1.4) were assigned to prenatal immune stimulation (LPS; n = 7; administered 2.5 µg/kg BW LPS i.m. at d 78 ± 1.8 of gestation) or saline treatment groups (CON; n = 7). From the 2 prenatal treatment groups, barrows (n = 17 LPS, 17 CON) were identified at weaning (21 ± 1.3 d of age) to subsequently receive a post-weaning LPS challenge. On d -7, barrows were transported to the USDA-ARS Livestock Issues Research Unit in Lubbock, TX where they were housed in individual pens in an environmentally controlled building with ad libitum access to water and a starter ration. On d -1, pigs were fitted with indwelling jugular catheters and subcutaneous temperature loggers. On d 0, pigs were challenged i.v. with LPS (10 µg/kg BW) and blood samples were collected at -2, 0, 1, 2, 4, 6, 8, 12, and 24 h relative to LPS challenge. Data were analyzed using PROC MIXED in SAS, specific for repeated measures. There was a treatment × time interaction for subcutaneous temperature (P &amp;lt; 0.01), where temperature increased more quickly at 1 and 2 h post-challenge in LPS compared with CON pigs. There was a tendency (P = 0.08) for less change in white blood cells, relative to baseline values, in LPS compared with CON pigs. There was a treatment × time interaction (P = 0.01) for lymphocyte concentrations where concentrations were reduced in LPS compared with CON pigs at 8 h post-challenge. There was also a treatment × time interaction (P = 0.01) for the change in eosinophil concentrations, where there was less change in eosinophil concentrations from 1 to 12h in LPS compared with CON pigs. There was a tendency (P = 0.08) for a treatment × time interaction for serum interleukin-6 (IL-6), where concentrations were reduced in LPS compared with CON pigs at 2h (P = 0.01) and tended to be reduced at 1 and 4h post-challenge (P ≤ 0.11). There was a tendency (P = 0.06) for a treatment × time interaction for IL-8, where concentrations were reduced in LPS compared with CON pigs at 1 and 4h (P ≤ 0.01) and tended to be reduced at 2h post-challenge (P = 0.06). Granulocyte macrophage colony stimulating factor and tumor necrosis factor-α tended (P ≤ 0.08) to be reduced in LPS compared with CON pigs. These data suggest that exposure to endotoxin in utero can influence the postnatal innate immune response to endotoxin. Thus, it is possible to influence the innate immune response of pigs in utero, which may serve as a method to positively influence postnatal immune responsiveness.

FLT3-Ligand Alone Enables the Identification and Maintenance in Vitro of Viable Quiescent Human Fetal Liver Hematopoietic Stem Cells with Maintenance of Their Function

Cells with sustained multi-lineage blood cell regenerative potential are referred to as hematopoietic stem cells (HSCs). Current findings indicate that they are responsible for lifelong blood production, are first detected within the first month of development in humans and are largely created prior to birth after which they expand their numbers in response to physiological demands for normal or enhanced blood cell output requirements. Interestingly, a number of the intrinsically determined functional properties of HSCs also change after birth. These include an apparent decline in their self-renewal potential. Thus, for example, in humans as in mice, cells with long-term (&amp;gt;6 month) HSC regenerative properties present in the developing fetal liver (FL) are a source of HSCs that possess a much higher in vivo regenerative capability than those from older donor sources, such as adult bone marrow or even cord blood (CB). Accordingly, it has been of longstanding interest to better understand the molecular regulation of this high regenerative capacity prevalent in fetal HSCs for potential future therapeutic as well as scientific exploitation. Here, we describe the results of experiments designed to answer the hypothesis that human FL HSCs with human-relevant self-renewal properties can be isolated as a quiescent CD49f+ subset of the GPI80+CD90+CD38-CD45RA-CD34+CD45+ population following their incubation in standard serum-free culture medium supplemented with FLT3-ligand (FLT3-L) alone. Initial experiments showed expression of the CD49f integrin on first trimester hFL cells selectively depleted cells able to produce colonies of granulocytes, macrophages or erythroid cells in standard 2-week methylcellulose cultures containing SCF, GM-CSF, IL6, IL3 and EPO. Conversely, expression of the CD49f integrin selectively enriched for cells with 12-week output capabilities in both growth factor (GF)-supplemented stromal co-cultures and in sublethally irradiated, transplanted immunodeficient NOD-Rag1 -/-IL2Rγc -/- W 41/41 (NRG-W) mice. Initial experiments designed to test the effect of multiple GF and small molecule additives on the maintenance over a 7-day period of this in vivo regenerative ability of the input FL HSC confirmed GPI80 expression to be a continuing positive selective phenotype. In addition, the result of transplant experiments showed that a 2-day incubation in FLT3-L alone maintained the 12-week serially transplantable activity of the HSCs (12-weeks/cycle) as fully equivalent to the unmanipulated input cells and significantly superior (P&amp;lt;0.05) to FLT3-L+IL3+SF (3GF) with or without addition of UM171, stemregenin, or eltrombopag. Interestingly, in vitro monitoring of GPI80+ cells showed FLT3-L alone maintained the survival of only 20% of the input GPI80+ cells compared to any of the 3GF-based conditions. In addition, the time to complete a first division and subsequent divisions of the input cells was delayed and prolonged, respectively, in the FLT3-L versus the 3GF conditions. Subsequent 7-day suspension cultures and 6-week GF-supplemented stromal co-cultures experiments have confirmed the existence of a G0 population within the HSC-enriched subset of human FL cells that best maintains their growth potential in FLT3-L alone. Together, these results demonstrate CD49f expression to be a pervasive marker of human HSCs throughout development and reveal the importance of different GF conditions to support the maintenance of viability and retention of self-renewal capability of human fetal HSCs in contrast to those required to activate/support a rapid initiation of cell division. These findings set the stage for future development of strategies to exploit human FL cells therapeutically and may also be critical to designing conditions that will best support the ex vivo maintenance of HSCs at and after birth.

The novel role of activating receptor KIR2DS5 in preeclampsia

Preeclampsia (PE) is a serious complication of pregnancy. Decidual natural killer (dNK) cells were reported to participate in the remodeling of spiral arteries through producing a group of cytokines, including granulocyte–macrophage colony stimulating factor (GM-CSF). KIR2DS5 is an activating receptor of NK cells that specifically recognizes HLA-C2 on trophoblasts. Currently, there are no reports regarding the precise mechanism of KIR2DS5 in PE. This study included 30 PE patients and 30 healthy pregnant women. We found that the expressions of KIR2DS5 were significantly lower in PE deciduae compared to those of healthy pregnancies. By transfecting knockdown and overexpression lentivirus vectors of KIR2DS5 into dNK cells isolated from deciduae of early pregnancy, we altered the KIR2DS5 expression level in dNK cells. Then, these dNK cells and trophoblast cell lines were co-cultured as trophoblast-dNK cells. In the trophoblast-dNK cells, we examined the influence of KIR2DS5 on the biological manifestations of trophoblasts. As anticipated, overexpression of KIR2DS5 could facilitate cell proliferation, migration, and invasion. Furthermore, increased expression of KIR2DS5 inhibited cell apoptosis and enhanced the progression of cells from theG1 to theS stage. Further mechanistic study demonstrated a positive relationship between KIR2DS5 and GM-CSF in trophoblast-dNK cells. Accordingly, our observations indicated that a decrease in KIR2DS5 could reduce the expression of GM-CSF via the JAK2/STAT5 pathway, resulting in the failure of the activated signal to be transmitted to dNK cells and ultimately leading to the occurrence of PE. KIR2DS5 may be a new contributor for the prediction and diagnosis of PE.

The interleukin-6 signal regulates orthodontic tooth movement and pain

Orthodontic tooth movement (OTM) is accomplished by controlling the mechanical loading onto the bone around the roots of target teeth. The precise orthodontic force induces osteoclastic bone resorption on the compression side and osteoblastic bone formation on the tension side of the alveolar bone. Orthodontic intervention causes inflammation in the periodontal ligament (PDL), which manifests as acute pain. Because inflammation is deeply connected to bone remodeling, it has been indicated that the inflammation after orthodontic intervention affects both the movement of teeth and generation of pain. However, the precise mechanisms underlying the immune regulation of OTM and the related pain are not well elucidated. Here, we found from the search of a public database that the interleukin (IL)-6 family of cytokines are highly expressed in the PDL by mechanical loading. The IL-6 signal was activated in the PDL after orthodontic intervention. The signal promoted OTM by inducing osteoclastic bone resorption. IL-6 was found to increase the number of osteoclasts by suppressing apoptosis and increasing their responsiveness to macrophage colony–stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). Furthermore, IL-6 signal was shown to elicit orthodontic pain by inducing neuroinflammation in the trigeminal ganglion (TG). Taken together, it was demonstrated that the IL-6 signal regulates tooth movement and pain during orthodontic treatment. It was also indicated that local blockade of the IL-6 signal is a promising therapeutic option in orthodontic treatment, targeting both tooth movement and pain.

Modulation of the immune response by the host defense peptide IDR-1002 in chicken hepatic cell culture

IDR-1002, a synthetic host defense peptide (HDP), appears to be a potential candidate for the treatment of bacterial infections and the consequent inflammatory response due to its potent immunomodulatory activity. This is of relevance to the emerging issue of antimicrobial resistance in the farming sector. In this study, the effects of IDR-1002 were investigated on a chicken hepatocyte‒non-parenchymal cell co-culture, and the results revealed that IDR-1002 had complex effects on the regulation of the hepatic innate immunity. IDR-1002 increased the levels of both RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) and Macrophage colony stimulating factor (M-CSF), suggesting the peptide plays a role in the modulation of macrophage differentiation, also reflected by the reduced concentrations of interleukin (IL)-6 and IL-10. The pro-inflammatory cytokine release triggered by the bacterial cell wall component lipoteichoic acid (LTA) was ameliorated by the concomitantly applied IDR-1002 based on the levels of IL-6, chicken chemotactic and angiogenic factor (CXCLi2) and interferon (IFN)-γ. Moreover, the production of nuclear factor erythroid 2-related factor 2 (Nrf2), an essential transcription factor in the antioxidant defense pathway, was increased after IDR-1002 exposure, while protein carbonyl (PC) levels were also elevated. These findings suggest that IDR-1002 affects the interplay of the cellular immune response and redox homeostasis, thus the peptide represents a promising tool in the treatment of bacterially induced inflammation in chickens.

Cross-Protection Conferred by Combined Vaccine Containing Infectious Bronchitis Virus Attenuated Massachusetts and Recombinant LaSota Virus Expressing Arkansas Spike.

We previously demonstrated that a prime-boost regime with an infectious bronchitis virus (IBV) Massachusetts (Mass)-type vaccine and recombinant LaSota virus (rLS) coexpressing IBV Arkansas (Ark)-type trimeric spike ectodomain (Se) and granulocyte macrophage colony stimulating factor (GMCSF) enhances heterologous protection against virulent Ark challenge. This study evaluates protection against Ark-type challenge conferred by administering the rLS/ArkSe.GMCSF and the attenuated Mass viruses mixed in the same vial as a combined vaccine. Chickens were vaccinated at day of hatch and challenged at 21 days of age with virulent Ark. Protection conferred by vaccination was assessed by respiratory signs, tracheal virus isolation as well as IBV RNA quantitation, and tracheal histomorphometry. Protection conferred by the combined vaccine was compared to protection induced by a commercial attenuated ArkDPI (Delmarva Poultry Industry) vaccine as well as by the attenuated Mass vaccine alone. Vaccination with the combined vaccine (rLS/ArkSe.GMCSF + Mass) as well as Mass alone provided significantly less protection against Ark challenge compared to the control using attenuated live ArkDPI vaccine. Only ArkDPI-vaccinated chickens exhibited "sterilizing immunity," i.e., no virus isolated from ≥10% of chickens after challenge. Chickens vaccinated with the combined vaccine rLS/ArkSe.GMCSF + Mass showed significantly less tracheal damage than birds vaccinated with the attenuated Mass vaccine alone. In addition, the combined vaccine also resulted in less virus isolation from the trachea. We concluded that the combined vaccine containing the recombinant virus and the attenuated Mass enhanced the cross-protective ability of the attenuated Mass vaccine against heterologous challenge.

Molecular characterization, gene expression and functional analysis of goldfish (Carassius auratus L.) macrophage colony stimulating factor 2.

Macrophage colony-stimulating factor 2 (MCSF-2) is an important cytokine that controls how cells of the monocyte/macrophage lineage proliferate, differentiate, and survive in vertebrates. Two isoforms of MCSF have been identified in fish, each exhibiting distinct gene organization and expression patterns. In this study, we investigated a goldfish MCSF-2 gene in terms of its immunomodulatory and functional properties. In this study, goldfish were acclimated for 3 weeks and sedated with TMS prior to handling. Two groups of fish were used for infection experiments, and tissues from healthy goldfish were collected for RNA isolation. cDNA synthesis was performed, and primers were designed based on transcriptome database sequences. Analysis of gfMCSF-2 sequences, including nucleotide and amino acid analysis, molecular mass prediction, and signal peptide prediction, was conducted. Real-time quantitative PCR (qPCR) was used to analyze gene expression levels, while goldfish head kidney leukocytes (HKLs) were isolated using standard protocols. The expression of gfMCSF-2 in activated HKLs was investigated, and recombinant goldfish MCSF-2 was expressed and purified. Western blot analysis, cell proliferation assays, and flow cytometric analysis of HKLs were performed. Gene expression analysis of transcription factors and pro-inflammatory cytokines in goldfish head kidney leukocytes exposed to rgMCSF-2 was conducted. Statistical analysis using one-way ANOVA and Dunnett's post hoc test was applied. We performed a comparative analysis of MCSF-1 and MCSF-2 at the protein and nucleotide levels using the Needleman-Wunsch algorithm. The results revealed significant differences between the two sequences, supporting the notion that they represent distinct genes rather than isoforms of the same gene. Sequence alignment demonstrated high sequence identity with MCSF-2 homologs from fish species, particularly C. carpio, which was supported by phylogenetic analysis. Expression analysis in various goldfish tissues demonstrated differential expression levels, with the spleen exhibiting the highest expression. In goldfish head kidney leukocytes, gfMCSF-2 expression was modulated by chemical stimuli and bacterial infection, with upregulation observed in response to lipopolysaccharide (LPS) and live Aeromonas hydrophila. Recombinant gfMCSF-2 (rgMCSF-2) was successfully expressed and purified, showing the ability to stimulate cell proliferation in HKLs. Flow cytometric analysis revealed that rgMCSF-2 induced differentiation of sorted leukocytes at a specific concentration. Moreover, rgMCSF-2 treatment upregulated TNFα and IL-1β mRNA levels and influenced the expression of transcription factors, such as MafB, GATA2, and cMyb, in a time-dependent manner. Collectively, by elucidating the effects of rgMCSF-2 on cell proliferation, differentiation, and the modulation of pro-inflammatory cytokines and transcription factors, our findings provided a comprehensive understanding of the potential mechanisms underlying gfMCSF-2-mediated immune regulation. These results contribute to the fundamental knowledge of MCSF-2 in teleosts and establish a foundation for further investigations on the role of gfMCSF-2 in fish immune responses.

RANKL-dependent osteoclast differentiation and gene expression in bone marrow-derived cells from adult mice is sexually dimorphic

Sex-specific differences in bone integrity and properties are associated with age as well as the number and activity of cells involved in bone remodeling. The aim of this study was to investigate sex-specific differences in adhesion, proliferation, and differentiation of mouse bone marrow derived cells into osteoclasts. The adherent fraction of bone marrow- derived cells from 12-week-old male and female C57BL/6J mice were assessed for their adhesion, proliferation, and receptor activator of nuclear factor κB (RANKL)-induced differentiation into osteoclasts. Female bone marrow derived macrophages (BMDMs) displayed higher adhesion and proliferation ratio upon macrophage colony stimulating factor (M-CSF) (day 0) and M-CSF + RANKL (day 4) treatment, respectively. On the contrary, male BMDMs differentiated more efficiently into osteoclasts upon RANKL-treatment compared to females (day 5). To further understand these sex-specific differences at the gene expression level, BMDMs treated with M-CSF (day 0) and M-CSF + RANKL (day 4), were assessed for their differential expression of genes through RNA sequencing. M-CSF treatment resulted in 1106 differentially expressed genes, while RANKL-treatment gave 473 differentially expressed genes. Integrin, adhesion, and proliferation-associated genes were elevated in the M-CSF-treated female BMDMs. RANKL-treatment further enhanced the expression of the proliferation- associated genes, and of genes associated with inhibition of osteoclast differentiation in the females, while RANK-signaling-associated genes were upregulated in males. In conclusion, BMDM adhesion, proliferation and differentiation into osteoclasts are sex-specific and may be directed by the PI3K-Akt signaling pathway for proliferation, and the colony stimulating factor 1-receptor and the RANKLsignaling pathway for the differentiation.

Decaffeinated coffee and green tea extract inhibit foam cell atherosclerosis by lowering inflammation and improving cholesterol influx/efflux balance through upregulation of PPARγ and miR-155

Background: Foam cells, the markers of early atherosclerosis and characterise the advanced atherosclerotic plaque, are stimulated by inflammation due to the excess of lipid in macrophages. The combination of decaffeinated coffee and green tea extract (DCGTE) has been suggested to have a role in foam cell inhibition. Objective: to investigate the role of DCGTE against foam cell formation, through modulation of the inflammation process and cholesterol metabolism in macrophage colony stimulating factor (M-CSF) and oxidized low-density lipoprotein (oxLDL)-exposed macrophages. Methods: Coffee and green tea were extracted by filtration and infusion respectively. Both of coffee and green tea underwent decaffeination using active carbon and blanching methods, respectively. Raw 264.7 Cells were administered with 160/160 and 320/320 μg/ml of DCGTE. Foam cell formation was observed using a light microscope after staining with Oil Red O (ORO), and quantified with ELISA. The expression of cluster differentiation 36 (CD36) ( lipid influx) and ATP binding cassette transporter A1 (ABCA1) ( cholesterol efflux) were determined through immunofluorescence. Tumor Necrosis Factor α (TNFα) and Interleukin10 (IL10) were quantified with ELISA. Peroxisome proliferator activated response γ (PPARγ) expression and activity were assessed with Polymerase Chain Reaction (PCR) and ELISA, respectively. The expression of microRNA 155 (miR-155) was examined using qPCR. Results: DCGTE at the above concentrations tended to reduce foam cell numbers, significantly inhibited lipid accumulation (p=0.000), reduced CD36 expression (p=0.000) and TNFα secretion (p=0.000) in Raw264.7 exposed to M-CSF 50 ng/ml and oxLDL 50 μg/ml. The PPARγ expression (p=0.000), PPARγ activity (p=0.001), ABCA1 (p=0.002), miR-155 relative expression (p=0.000), and IL10 production (p=0.000) also increased in DCGTE groups. Conclusion: DCGTE lowered foam cell possibly through attenuation of the inflammatory process and improvement of lipid/efflux mechanisms via upregulation of PPARγ and miR-155 in M-CSF and oxLDL-stimulated Raw264.7 cells. Our results suggest DCGTE might help to prevent atherosclerosis-based diseases.