M-CSF Stimulation Research Articles

Withholding of M-CSF Supplement Reprograms Macrophages to M2-Like via Endogenous CSF-1 Activation.

Macrophage colony-stimulating factor (M-CSF or CSF-1) is known to have a broad range of actions on myeloid cells maturation, including the regulation of macrophage differentiation, proliferation and survival. Macrophages generated by M-CSF stimulus have been proposed to be alternatively activated or M2 phenotype. M-CSF is commonly overexpressed by tumors and is also known to enhance tumor growth and aggressiveness via stimulating pro-tumor activities of tumor-associated macrophages (TAMs). Currently, inhibition of CSF-1/CSF-1R interaction by therapeutic antibody to deplete TAMs and their pro-tumor functions is becoming a prevalent strategy in cancer therapy. However, its antitumor activity shows a limited single-agent effect. Therefore, macrophages in response to M-CSF interruption are pending for further investigation. To achieve this study, bone marrow derived macrophages were generated in vitro by M-CSF stimulation for 7 days and then continuously grown until day 21 in M-CSF absence. A selective pressure for cell survival was initiated after withdrawal of M-CSF. The surviving cells were more prone to M2-like phenotype, even after receiving interleukin-4 (IL-4) stimulation. The transcriptome analysis unveiled that endogenous CSF-1 level was dramatically up-regulated and numerous genes downstream to CSF-1 covering tumor necrosis factor (TNF), ras-related protein 1 (Rap1) and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway were significantly modulated, especially for proliferation, migration and adhesion. Moreover, the phenomenal increase of miR-21-5p and genes related to pro-tumor activity were observed in parallel. In summary, withholding of CSF-1/CSF-1R interaction would rather augment than suspend the M-CSF-driven pro-tumor activities of M2 macrophages in a long run.

Knockout of purinergic receptor Y13 (P2Y13) results in an improved outcome of metabolic syndrome in mice

Abstract Metabolic syndrome (MetS) clusters the main risk factors for cardiovascular diseases (CVDs) and endocrine dysfunction. The pathomechanism of MetS is described as local death of hypertrophic adipocytes releasing danger-associated molecular patterns (DAMPs) such as nucleotides (e.g. ADP). This promotes a long term inflammation of adipose tissue via activation of purinergic receptors with a gradual shift towards a pro-inflammatory environment. The ADP receptor P2Y13 is both described in metabolic and immunological processes. These combined characteristics make the P2Y13 an interesting candidate to investigate its role in MetS. Nevertheless, the role of P2Y13R in the pathogenesis of MetS is currently unknown and shall be analyzed in this study. BMDM isolation and differentiation to Mϕ using M-CSF and subsequent stimulation with medium, LPS and IFNγ or IL4; Expression was quantified using Taqman. Male C57Bl6/J wild-type (WT) and P2Y13-deficient (KO) mice were fed a HFD for 20 weeks; body weight and food consumption were recorded weekly. GTT, ITT and metabolic cages were performed after 15 weeks with euthanization after 20 weeks. In order to distinguish the effect of hematopoetic or somatic cells, mice were lethally irradiated with 9.4Gy and reconstituted with donor bone-marrow (BM) via tail vein injection. We observed a unique expression of P2Y13R on pro-inflammatory M1 Mϕ. After HFD feeding KO mice showed higher O2 consumption compared to WT mice (AUC of O2 consumption on 2nd day= KO:61620±2261mL/kg vs WT: 53830±916mL/kg, p<0.05). Although KO mice consumed more food compared to WT littermates (KO:5.7±0.5g/d vs WT:3.1±0.1g/d, p<0.0001), they showed significantly decelerated weight gain (e.g.week 15→KO:147,292±5,26% vs WT:180.8±15.9%, p<0.05). Obese KO animals outperformed obese WT littermates in a peritoneal glucose tolerance test (GTT) (2h post-injection (post-i.) →KO:273.7±46.3mg/dL vs WT: 555.0±40.8mg/dL, p<0.05). KO mice on HFD were protected from developing a fatty liver. HFD KO mice receiving WT BM show accelerated weight gain compared to KO mice receiving KO BM (e.g.week 10 WT in KO: 111.2±2.2% vs KO in KO: 102.2±1.2%, p<0.05). In the GTT irradiated KO mice either receiving KO or WT BM are protected from HFD induced impaired glucose homeostasis (45min post-i.; KO→KO:222.1±21.2mg/dL vs WT→KO:232.8±15.9mg/dL vs WT on chow diet:240.4±18.6mg/dL). Contrary, WT mice receiving KO or WT BM developed a glucose resistance comparable to non-irradiated WT mice (45min post-i.; WT→WT:423.8±61.7mg/dL vs KO→WT:434.3±51.1 vs WT on HFD:574.4±7.9). P2Y13 KO improves the outcome of MetS in mice with improved glucose homeostasis, decelerated weight gain, no fatty liver development and better metabolic turnover. BM transplantation experiments suggest a somatic component as possible explanation of these observations. Given these beneficial metabolic effects, we hypothesize that antagonization of P2Y13R could be a promising therapeutic tool in the field of MetS. Funding Acknowledgement Type of funding source: None

5221Knockout of purinergic receptor Y13 (P2Y13) results in an improved outcome in metabolic syndrome in mice

Abstract Background Metabolic syndrome clusters the main risk factors for cardiovascular diseases and endocrine dysfunction. Novel studies show that the important underlying mechanism is a long term inflammation of adipose tissue with a gradual shift from the anti-inflammatory M2 towards the pro-inflammatory M1 macrophages (pathognomonic). Massive hypertrophy induces adipocyte death, which releases DAMPs like nucleotides recognized by purinergic receptors orchestrating ongoing inflammatory process. Interestingly, we found Gi coupled P2Y13R only expressed on M1 macrophages, not in M0 (unstimulated) and M2 macrophages. Nevertheless, the role of P2Y13R in the immune system and especially macrophages is currently unknown. Given its pivotal role in central metabolic processes (P2Y13R has been described in insulin secretory signalling) together with its unique expression in its pathognomonic inflammatory macrophage subtype makes it an interesting candidate to investigate its role in metabolic syndrome. Purpose Due to the unique expression of P2Y13R on M1 macrophages we hypothesise an improved outcome in a high-fat diet induced metabolic syndrome by interfering with the P2Y13 signalling cascade. Methods BMDM differentiation to macrophages using M-CSF and subsequent stimulation with medium (M0), LPS and IFNγ (M1) or IL4 (M2); Expression of P2Rs quantified using Taqman qPCR. Male C57Bl6/J wild-type (WT) and P2Y13-deficient (KO) mice were fed with a high-fat diet (HFD) for 20 weeks; On week 15 we performed the ITT, on week 16 the GTT. Metabolic performance was monitored by metabolic caging. Results We observed a unique expression of P2Y13R on M1 macrophages. Adult P2Y13-deficient mice showed a higher O2 consumption compared to adult C57Bl6/J wild-type mice (AUC of O2 consumption 2nd day= KO: 61620±2261mL/kg vs WT: 53830±916.1mL/kg, p=0.0331). Although both P2Y13−/− mice and WT littermates consumed comparable amount of food (daily food intake per mouse → KO: 3.97±0.25g vs WT: 3.76±0.18g), P2Y13 deficient animals showed significantly decelerated weight gain (e.g. on week 15 → KO: 142±2% (n=10) vs WT: 198±5% (n=10), p<0.0001). Obese P2Y13−/− animals outperformed obese WT littermates in a peritoneal glucose tolerance test (2h after glucose injection → KO: 272.9±21.0 mg/dL (n=10) vs WT: 532.6±21.2 (n=10) mg/dL, p<0.0001). There was no difference in the cell amount of stromal vascular fraction cells. Conclusion Global P2Y13 deficiency leads to an improved outcome in metabolic syndrome with an increased protection against developing an insulin resistance as shown through an improved glucose tolerance and basal glucose levels, a decelerated weight gain despite comparable food consumption and a better metabolic turnover. Observing these beneficial metabolic improvements, we hypothesise that antagonization of P2Y13R could be a promising therapeutic target in the field of metabolic syndrome.

Characterization of the Angiogenic Potential of Human Regulatory Macrophages (Mreg) after Ischemia/Reperfusion Injury In Vitro.

Ischemia/reperfusion- (I/R-) induced organ damage represents one of the main causes of death worldwide, and new strategies to reduce I/R injury are urgently needed. We have shown that programmable cells of monocytic origin (PCMO) respond to I/R with the release of angiogenic mediators and that transplantation of PCMO results in increased neovascularization. Human regulatory macrophages (Mreg), which are also of monocytic origin, have been successfully employed in clinical transplantation studies due to their immunomodulatory properties. Here, we investigated whether Mreg also possess angiogenic potential in vitro and could represent a treatment option for I/R-associated illnesses. Mreg were differentiated using peripheral blood monocytes from different donors (N = 14) by incubation with M-CSF and human AB serum and stimulation with INF-gamma. Mreg cultures were subjected to 3 h of hypoxia and 24 h of reoxygenation (resembling I/R) or the respective nonischemic control. Cellular resilience, expression of pluripotency markers, secretion of angiogenic proteins, and influence on endothelial tube formation as a surrogate marker for angiogenesis were investigated. Mreg showed resilience against I/R that did not lead to increased cell damage. Mreg express DHRS9 as well as IDO and display a moderate to low expression pattern of several pluripotency genes (e.g., NANOG, OCT-4, and SOX2). I/R resulted in an upregulation of IDO (p < 0.001) while C-MYC and KLF4 were downregulated (p < 0.001 and p < 0.05). Proteome profiling revealed the secretion of numerous angiogenic proteins by Mreg of which several were strongly upregulated by I/R (e.g., MIP-1alpha, 19.9-fold; GM-CSF, 19.2-fold; PTX3, 5.8-fold; IL-1β, 5.2-fold; and MCP-1, 4.7-fold). The angiogenic potential of supernatants from Mreg subjected to I/R remains inconclusive. While Mreg supernatants from 3 donors induced tube formation, 2 supernatants were not effective. We suggest that Mreg may prove beneficial as a cell therapy-based treatment option for I/R-associated illnesses. However, donor characteristics seem to crucially influence the effectiveness of Mreg treatment.

MiR-125a-5p promotes osteoclastogenesis by targeting TNFRSF1B

AimTo investigate the dysregulation of microRNAs (miRNAs) during the differentiation of osteoclasts and the precise roles of miR-125a-5p in the differentiation of osteoclasts.MethodsThe cell model of RAW 264.7 osteoclast precursor cell differentiation induced by RANKL plus M-CSF stimulation was established. During the early stage of osteoclast differentiation, miRNA expression profiles were detected using the biochip technique and analyzed by cluster analysis. TargetScan, miRTarBase and miRDB database analysis was applied to find the key target genes of miR-125a-5p. A dual luciferase experiment was conducted to identify the direct target of miR-125a-5p. MiR-125a-5p mimic transfection and anti-miR-125-5p treatment were conducted to verify the role of miR-125q-5p in osteoclast differentiation. The levels of triiodothyronine receptor auxiliary protein (TRAP), matrix metallopeptidase 2 (MMP-2), MMP-9 and cathepsin K were analyzed by qRT-PCR and western blot assay. The expression levels of MMP-2 and MMP-9 were determined using western blotting and immunofluorescence assay. The migration and invasion of RAW 264.7 cells were assessed by wound healing and Transwell invasion assays. The proliferation of RAW 264.7 osteoclast precursor cells was detected using MTT assay.ResultsThere were 44 microRNAs differently expressed during the differentiation of RAW 264.7 osteoclast precursor cells into osteoclasts, 35 of which were up-regulated and 9 were down-regulated. By luciferase reporter assay, it was confirmed that the TNF receptor superfamily member 1B gene (TNFRSF1B) was the target gene of miR-125a-5p. Up-regulation of miR-125a-5p inhibited TNFRSF1B protein expression and promoted osteoclast differentiation whereas down-regulation of miR-125a-5p caused completely opposite results.ConclusionsIn conclusion, overexpression of miR-125a-5p suppresses the expression of TNFRSF1B and promotes osteoclast differentiation. These results reveal the crucial role of miR-125a-5p in the differentiation of osteoclasts.

Upregulation of miR-196b-5p attenuates BCG uptake via targeting SOCS3 and activating STAT3 in macrophages from patients with long-term cigarette smoking-related active pulmonary tuberculosis

BackgroundCigarette smoking (CS) triggers an intense and harmful inflammatory response in lungs mediated by alveolar and blood macrophages, monocytes, and neutrophils and is closely associated with prevalence of tuberculosis (TB). The risk of death in patients with long-term cigarette smoking-related pulmonary tuberculosis (LCS-PTB) is approximately 4.5 times higher than those with nonsmoking pulmonary tuberculosis (N-PTB). However, the mechanisms underlying the harmful inflammatory responses in the setting of LCS-PTB have not been well documented.Methods28 cases LCS-PTB patients, 22 cases N-PTB patients and 20 cases healthy volunteers were enrolled in this study. Monocytes were isolated from peripheral blood mononuclear cells. Differentiated human MDM and U937 cell were prepared with M-CSF and PMA stimulation, respectively. The miR-196b-5p, STAT1, STAT3, STAT4, STAT5A, STAT5B, STAT6, SOCS1 and SOCS3 mRNA expression were detected by qRT-PCR. Western blot was performed according to SOCS1, SOCS3, and pSTAT3 expression. The mycobacterial uptake by MDMs from different groups of patients after Bacillus Calmette–Guérin (BCG) infection and agomir-196b-5p or antagomir-196b-5p transfection were used by flow cytometry analysis. Human IL-6, IL-10 and TNF-α levels on the plasma and cell culture supernatant samples were measured using ELISA. For dual-luciferase reporter assay, the SOCS3 3′-UTR segments, containing the binding elements of miR-196b-5p or its mutant versions were synthesized as sense and antisense linkers.ResultsIn this study, we found that IL-6, TNF-α production, SOCS3 mRNA expression were downregulated, while miR-196b-5p and STAT3 mRNA expression were upregulated in monocytes from LCS-PTB patients as compared to N-PTB patients. Meanwhile, we demonstrated that miR-196b-5p could target SOCS3 and activate STAT3 signaling pathway, which may possibly contribute to attenuation of BCG uptake and decrease in IL-6 and TNF-α production in macrophages.ConclusionsOur findings revealed that CS exposure regulates inflammatory responses in monocyte/macrophages from LCS-PTB patients via upregulating miR-196b-5p, and further understanding of the specific role of miR-196b-5p in inflammatory responses mightfacilitate elucidating the pathogenesis of LCS-PTB, thus leading to the development of new therapeutic strategies for PTB patients with long-term cigarette smoking.

Immunophenotyping of chemokine receptor profile on osteoclast progenitors may help reveal mechanisms of their increased migration in collagen-induced arthritis

Abstract Osteoclasts, specialized bone resorbing cells of hematopoietic origin, become overactive in rheumatoid arthritis, leading to bone loss and joint destruction. Osteoclast progenitor cells (OCP) arise from myeloid precursors of monocyte/macrophage lineage and are found in the bone marrow and among circulatory monocytes. Under inflammatory conditions subpopulations of OCPs undergo through functional and phenotypic changes responsible for their increased migration to inflamed sites and osteoresorptive activity. In our study, we have been investigating frequencies of distinct OCP subsets and their expression of chemokine receptors in circulation and periarticular bone marrow (PBM) of C57BL/6 mice with collagen-induced arthritis (CIA). We found that both hematopoietic lymphoid-negative CD11b+CD115+ and CD11b−/loCD115+ subsets possess osteoclastogenic activity and are enlarged in blood and PBM of affected joints in CIA. OCP subsets substantially expressed CCR2 and CX3CR1, but had low expression of CCR5, CCR9 and CXCR4, with no difference between control and CIA group. Concurrently CCL2 serum levels were significantly increased in CIA, whereas CX3CL1 levels were comparable to control. In an in vitro migration assay, peripheral blood leukocytes from CIA mice after M-CSF and RANKL stimulation increasingly migrated toward CCL2 and CCL5 chemotactic gradients. Intravascular in vivo staining demonstrated increased recirculation of CD45+CD11b+ cells through areas of PBM affected by arthritis. These results suggest that highly induced OCP subpopulations in CIA migrate to inflamed joints due to chemotaxis and therefore blocking of chemokine signals may reduce osteoclast activity in arthritis.

Calvariae-Resident Osteomacs That Are Phenotypically and Functionally Distinct from Marrow-Derived Macrophages Interact with Megakaryocytes to Regulate Hematopoietic Stem Cell Function

Maintenance of stem cell function is an orchestrated event requiring the participation of multiple cell types within the hematopoietic niche. Precise networking between hematopoietic stem cells (HSC) and these cell types is critical for the maintenance of the stem cell pool. Evidence is accumulating that multiple cell types cooperate to collectively maintain HSC function in the hematopoietic niche. We report here a detailed characterization of calvariae-resident osteomacs (OM) and outline how these cells require cooperation from megakaryocytes (MK) to sustain HSC function. We also describe in detail discriminating phenotypic and functional properties that clearly distinguish OM from marrow-derived macrophages (Mϕ).Osteomacs, identified as CD45+F4/80+ cells, were easily detectable in calvarial cell (CC) preparations (3-5% of total CC) collected by the enzymatic digestion of calvariae from 2d-old pups. To assess the effect of MK, a known regulator of osteoblast (OB) proliferation and differentiation, on OM, we performed co-cultures using CC and MK prepared from fetal liver. In the absence of MK, OM did not increase in numbers over a period of 5 days in culture and remained approximately 5% of total cultured cells. However, in the presence of MK, OM significantly increased to become between 25% and 30% of total cells demonstrating that MK regulate OM proliferation. Clonogenic assays established that OM support hematopoiesis enhancing activity of OB and that this activity can be upregulated by MK. Interestingly, marrow-derived Mϕ were unable to mediate the same hematopoiesis enhancing activity regardless of whether MK were present in the co-culture or not. These results were validated via primary and secondary transplantations in lethally irradiated hosts whereby the highest repopulating potential was observed among marrow-derived LSK cells co-cultured for 5 days with a mixture of OB, OM, and MK. Using eight surface markers and flow cytometric analysis, we established that although marrow-derived Mϕ and OM share many phenotypic similarities (CD45, F4/80, CD68, CD11b, Mac2, and GR-1), only OM expressed MCSFR and CD166, thus providing a distinct and unique profile for these cells.To assess changes in pathway activation between resting and MK-activated OM, we performed single cell genomic analysis. This approach detected the upregulation of several canonical pathways important in HSC maintenance such as Ephrin receptor signaling, PDGF signaling, and leukocyte extravasation signaling in MK-stimulated OM. Single cell genomic analysis between CC-derived OM and marrow-derived Mϕ (isolated from each tissue as CD45+F4/80+ cells) revealed 39 genes to be significantly different between the two cell types. Strikingly, many genes such as IGF1, KITL and NOTCH2 that have previously been implicated in HSC regulation were upregulated in OM. MCSFR1 a known regulator of proliferation, differentiation and survival of Mϕ was also upregulated in OM corroborating the data previously collected from flow cytometric analyses. However, OM did not respond to exogenous MCSF stimulation suggesting that MCSF alone is not sufficient to induce OM proliferation or that direct contact with MK is required for induction of proliferation. To investigate changes at the protein translational level, we examined both cell types using CyTOF and a panel of 24 surface and intracellular antibodies. The surface marker CD169 which was previously associated with HSC retention when present on cellular components of the hematopoietic niche was expressed on OM but not on Mϕ. Intriguingly, OM expressed both CD86 and CD206 which are known M1 and M2 Mϕ markers, respectively. TNF-α, TIMP2, FGF2 and MCP1 which are known HSC regulators were also upregulated in OM. Finally, the majority of OM expressed embigin and IL-18, both of which have been implicated in the maintenance of HSC function.These data demonstrate that although bone-associated OM share many properties with marrow-derived Mϕ, they are phenotypically and functionally distinct and are critical for the maintenance of HSC function. Furthermore, the function of OM, and consequently that of the two components of CC, namely OB and OM, is significantly augmented by interactions with MK demonstrating that the crosstalk between OM, OB and MK form a novel network in supporting HSC function. DisclosuresNo relevant conflicts of interest to declare.

Distinctive and selective route of PI3K/PKCα-PKCδ/RhoA-Rac1 signaling in osteoclastic cell migration

Cell migration during specialized stages of osteoclast precursors, mononuclear preosteoclasts, and multinucleated mature osteoclasts remain uncertain. M-CSF- and osteopontin-induced osteoclastic cell migration was inhibited by function-blocking monoclonal antibodies specific to the integrin αv and β3 subunits, suggesting that integrin αvβ3 mediates migratory signaling induced by M-CSF and osteopontin. M-CSF and osteopontin stimulation was shown to regulate two branched signaling processes, PI3K/PKCα/RhoA axis and PI3K/PKCδ/Rac1 axis. Interestingly, inactivation of RhoA or Rac1 blocked preosteoclast and mature osteoclast migration but not osteoclast precursor migration in a transwell-based cell migration assay. Moreover, the inhibitory effect on preosteoclast and mature osteoclast migration induced by Rac1 inactivation was more effective than that by RhoA inactivation. Collectively, our findings suggest that osteoclast precursor migration depends on PI3K/PKCα-PKCδ signaling mediated via integrin αvβ3 bypassing RhoA and Rac1, whereas preosteoclast and mature osteoclast migration relies on PI3K/PKCα-PKCδ/RhoA-Rac1 axis signaling mediated via integrin αvβ3 with increased dependency on PKCδ/Rac1 signaling route as differentiation progresses.

A non-canonical function of telomerase RNA in the regulation of developmental myelopoiesis in zebrafish

Dyskeratosis congenita (DC) is an inherited disorder with mutations affecting telomerase or telomeric proteins. DC patients usually die of bone marrow failure. Here we show that genetic depletion of the telomerase RNA component (TR) in the zebrafish results in impaired myelopoiesis, despite normal development of haematopoietic stem cells (HSCs). The neutropenia caused by TR depletion is independent of telomere length and telomerase activity. Genetic analysis shows that TR modulates the myeloid-erythroid fate decision by controlling the levels of the master myeloid and erythroid transcription factors spi1 and gata1, respectively. The alteration in spi1 and gata1 levels occurs through stimulation of gcsf and mcsf. Our model of TR deficiency in the zebrafish illuminates the non-canonical roles of TR, and could establish therapeutic targets for DC.

In Vitro Generation Of Osteoclasts From Interleukin-3 (IL-3)-Dependent Mouse Hematopoietic Cells

ObjectiveThis work aims to develop a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells Methods1. Here, we describe an alternative method for in vitro generation of osteoclasts, which involves the use of interleukin (IL)-3-dependent murine bone marrow cells. Bone marrow cells, isolated from 6- to 8-week old C57BL/6 were cultured in α-MEM containing 10% FBS in tissue culture dishes overnight to remove stromal cells. Then, non-adherent bone marrow cells were harvested and continued in α-MEM containing 10% FBS without (control) or with IL-3 (1 ng/ml) for 6 days. While no cells survived in the control culture after the 6-day culturing, the IL-3-treated culture gave rise to a significant number of surviving cells. These IL-3-depedent cells were capable of differentiating to osteoclasts in response to M-CSF and RANKL stimulation. Moreover, these IL-3-dependent cells can be further expanded by plating them in non-treated plastic dishes followed with M-CSF treatment; they continued to survive and proliferate in non-treated plastic dishes in the presence of M-CSF for up to 4 days. After 4-day M-CSF treatment, these cells can be lifted by EDTA, and they were still able to differentiate into osteoclasts upon subsequent stimulation of M-CSF and RANKL.2. We performed the in vitro bone resorption assay, Semiquantitative Reverse Transcription (RT)-PCR, Western Analysis, Infection of Murine Bone Marrow Cells (BMCs) to test whether the osteoclasts generated from IL-3-dependent murine bone marrow cells are different from the osteoclasts generated from traditional method. Results1. IL-3 can maintain the survival of murine bone marrow cells for up to 6 days and these cells still keep their capacity to generate osteoclasts. The capacity of IL-3-dependent cells to form osteoclasts decreases with time of IL-3 treatment and IL-3 dependent cells can be further expanded by M-CSF without significant loss of the osteoclastogenic potential.2. IL-3-dependent cells can form functional osteoclasts. RANKL induces the expression of osteoclast genes in IL-3-dependent cells. RANKL activates some of RANK signaling pathways in IL-3-dependent cells. Importantly, we found that IL-3 dependent murine bone marrow cells can be infected by retrovirus encoding GFP. Conclusions1) We have developed a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells.2) IL-3-dependent cells can be infected by retrovirus, permitting further experimental manipulations to express or knock down genes in IL-3-dependent cells for studying the molecular mechanism controlling differentiation and proliferation of osteoclast precursors or delineating molecular events in early osteoclastogenesis. Disclosures:No relevant conflicts of interest to declare.

M-CSF cooperating with NFκB induces macrophage transformation from M1 to M2 by upregulating c-Jun

Increasing evidence suggests tumor-associated macrophages (TAMs) are polarized M2 subtype of macrophage that exerts pro-tumor effects and promote the malignancy of some cancers, but the concrete mechanism is not well defined. Our previous research exhibited that proto-oncogene AP-1 regulated IL-6 expression in macrophages and promoted the formation of M2 macrophages. In this study, we investigate whether extra-cellular stimulus M-CSF help this process or nuclear factor NFκB has a synergistic role in the activation state of polarized M2 subtype of macrophage. RAW 264.7 macrophage and 4T1 mouse breast cancer cells were co-cultured to reconstruct tumor microenvironment. Being co-cultured with 4T1 or its supernatant, the expression of c-Jun, the member of AP-1 family, has a dramatically increase both on the level of gene and on the protein in RAW 264.7 macrophages, but the expression of c-Fos does not increase neither on the level of gene nor on the protein. After co-cultured with 4T1, RAW 264.7 has a higher consumption of M-CSF than RAW 264.7 macrophages alone. With the stimulation of M-CSF, the mRNA of c-Jun increased significantly, but decreased remarkably after adding the anti-M-CSF. And at the same time, p50, the member of NFκB family, has a similar tendency to c-Jun. WB results suggest that with the stimulation of M-CSF, p-Jun in nuclear increases heavily but decreases after the neutralizing antibody added. Coimmunoprecipitation and immunoblotting techniques confirmed that c-Jun and p50 NFκB coprecipitated, and c-Jun protein expression is properly enhanced with rM-CSF effect. In conclusion, M-CSF induces macrophage transformation by upregulating c-Jun with a certain synergy of NFκB. Our study may present a novel therapeutic strategy against cancer.

P36 Hydrogen sulfide inhibits RANKL-induced ROS production and osteoclast differentiation

Osteoclasts (OC), the bone resorbing cells, are multinucleated cells arising from the fusion of circulating monocytic precursors. Once differentiated, they settle into bone and break down bone mineral matrix, thereby playing a vital role in physiological bone turnover.In post-menopausal osteoporosis or in inflammatory bone disease, OC are activated leading to an excess bone erosion which in not balanced by an equal bone formation by osteoblasts, a process that leads to a loss of bone mass, increased bone fragility and fracture risk. The rate of monocyte differentiation into OC has been shown to be a critical step in pathological bone erosion.Here we sought to investigate whether hydrogen sulfide affect OC differentiation and function. Human osteoclast precursors were isolated from peripheral blood obtained from healthy donors by immunomagnetic positive selection of CD11b+ cells. Mature OC were obtained in vitro by culturing CD11b+ cells for 8–10 days in the presence of M-CSF (10 ng/ml) and RANKL (75 ng/ml). The effect of hydrogen sulfide was evaluated by treating the cells with NaHS in the range of concentrations between 50 and 300 μg/ml.We first assessed whether NaHS affect cell survival and proliferation. LDH activity assay revealed no cytotoxic effect of NaHS at 50, 10, 200 or 300 μg/ml at 24 h after stimulation, while 72 h after stimulation the concentration of 300 μg/ml induced a 25% increase in cell death. Cell proliferation was evaluated by 3 H-timidine uptake: consistent with citotoxicity data, NaHS did not affect the proliferation of CD11b+ cells in response to M-CSF stimulation up to a concentration of 200 μg/ml. Evaluation of apoptosis by Annexin V/PI further confirmed that NaHS did not induced cell death at 50, 100 or 200 μg/ml while induced a 30% increase of apoptosis at the concentration of 300 μg/ml. To assess the effect of NaHS on OC differentiation, TRAP + mature OC were counted at the end of the culture. NaHS dose-dependently inhibited OC formation, leading to a significant decrease in OC number starting a 100 μg/ml. Functional Pit Assay confirmed that NaHS significantly inhibits the ability of OC to break down a bone-like mineral matrix. Attesting to specificity, similar data were obtained in the presence of the H 2 S-donor GYY4137. Therefore, hydrogen sulfide inhibits OC differentiation and function in a range of concentration which does not affect cell survival. As RANKL signaling is critically dependent on ROS generation in OC progenitor cells, we evaluated the effect of NaHS on RANKL-dependent ROS generation by Dichlorofluorescein staining. As expected, RANKL (100 μg/ml, 30 min) induced a significant production of intracellular ROS in OC precursors cells; however, NaHS (200 μg/ml), significantly inhibited RANKL-induced ROS generation.In conclusion, Hydrogen sulfide dose-dependently prevents OC differentiation and function and this effect is associated to a significant inhibition of RANKL-induced ROS production. These findings suggest a possible therapeutical role for hydrogen sulfide donors in the treatment of pathological bone loss.

Tumor Associated Macrophage × Cancer Cell Hybrids May Acquire Cancer Stem Cell Properties in Breast Cancer

Breast cancer is one of the most frequently diagnosed cancers among women, and metastasis makes it lethal. Tumor-associated macrophages (TAMs) that acquire an alternatively activated macrophage (M2) phenotype may promote metastasis. However, the underlying mechanisms are still elusive. Here, we examined how TAMs interact with breast cancer cells to promote metastasis. Immunohistochemistry was used to examine the expression of the M2-specific antigen CD163 in paraffin-embedded mammary carcinoma blocks to explore fusion events in breast cancer patients. U937 cells were used as a substitute for human monocytes, and these cells differentiated into M2 macrophages following phorbol 12-myristate 13-acetate (PMA) and M-CSF stimulation. M2 macrophages and the breast cancer cell lines MCF-7 and MDA-MB-231 fused in the presence of 50% polyethylene glycol. Hybrids were isolated by fluorescence-activated cell sorting, and the relevant cell biological properties were compared with their parental counterparts. Breast cancer stem cell (BCSC)-related markers were quantified by immunofluorescence staining, RT-PCR, quantitative RT-PCR and/or western blotting. The tumor-initiating and metastatic capacities of the hybrids and their parental counterparts were assessed in NOD/SCID mice. We found that the CD163 expression rate in breast cancer tissues varied significantly and correlated with estrogen receptor status (p<0.05). The fusion efficiency of either breast cancer cell line with M2 macrophages ranged from 1.81 to 6.47% in the presence of PEG, and no significant difference was observed between the breast cancer cell lines used (p>0.05). Characterization of the fusion hybrids revealed a more aggressive phenotype, including increased migration, invasion and tumorigenicity, but reduced proliferative ability, compared with the parental lines. The hybrids also gained a CD44+CD24−/low phenotype and over-expressed epithelial-mesenchymal transition-associated genes. These results indicate that TAMs may promote breast cancer metastasis through cell fusion, and the hybrids may gain a BCSC phenotype.

Absence of MCP‐1 leads to elevated bone mass via impaired actin ring formation

Monocyte chemoattractant protein-1 (MCP-1) is associated with various inflammatory diseases involving bone loss, and is expressed along with its receptor by bone marrow-derived macrophages (BMM), which are osteoclast (OC) precursors. To investigate the role of MCP-1 in bone remodeling, we compared MCP-1-knockout (KO) mice with wild-type (WT) mice. The absence of MCP-1 increased bone mass and lowered serum collagen type I fragments (CTX-1) and TRACP 5b, but had no significant effect on the N-terminal propeptide of type I procollagen, suggesting that OCs are primarily responsible for the bone phenotype observed in the absence of MCP-1. MCP-1 deficiency resulted in reduced numbers and activity of OCs in vitro. It also led to a reduced level of c-Fms and receptor activator of nuclear factor-κB receptor and impaired actin ring formation. Activation of ERK, Akt, Rac1, and Rho upon M-CSF stimulation was also reduced and our evidence suggests that the aberrant actin ring formation was partly due to reduced activation of these molecules. Our findings point to a role of osteoclast MCP-1 in regulating bone remodeling. The higher bone mass in the femurs of MCP-1-KO mice could be, at least in part, due to decreased osteoclastogenesis and bone resorption resulting from aberrant M-CSF signaling in OCs.

Association of Oxidative Stress with Postmenopausal Osteoporosis and the Effects of Hydrogen Peroxide on Osteoclast Formation in Human Bone Marrow Cell Cultures

It has been suggested that oxidative stress is associated with the pathogenesis of osteoporosis. The objective of this study was to explore the association between a marker of oxidative stress and either bone turnover markers or bone mineral density (BMD) in postmenopausal women. In addition, the effects of oxidative stress on the formation of osteoclasts in human bone marrow cell culture were examined. We performed a cross-sectional analysis in healthy postmenopausal women aged 60-78 years (n = 135, 68.2 +/- 4.9). Oxidative stress was evaluated in the serum by measuring 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels. The biochemical markers of bone turnover and areal BMD were measured in all participants. Multivariate linear regression analysis revealed a negative association between 8-OH-dG levels and BMD of the lumbar spine, total hip, femoral neck, and trochanter and positive association with type I collagen C-telopeptide (ICTP) levels. The odds ratio of 8-OH-dG for osteoporosis was 1.54 (1.14-2.31, P = 0.003). In cultures of primary human marrow cells, H2O2 caused concentration-dependent activation of TRAP-positive multinucleated giant cells. H2O2 also increased the area of pits per osteoclast activity assay substrate. RT-PCR showed that H2O2 stimulated the expression of M-CSF and RANKL and increased the RANKL/OPG ratio. The data support the view that oxidative stress is associated with increased bone resorption and low bone mass in otherwise healthy women. In addition, RANKL and M-CSF stimulation induced by oxidative stress may participate in osteoclastogenesis in human bone.

Adaptor protein Lnk inhibits c-Fms-mediated macrophage function

The M-CSFR (c-Fms) participates in proliferation, differentiation, and survival of macrophages and is involved in the regulation of distinct macrophage functions. Interaction with the ligand M-CSF results in phosphorylation of tyrosine residues on c-Fms, thereby creating binding sites for molecules containing SH2 domains. Lnk is a SH2 domain adaptor protein that negatively regulates hematopoietic cytokine receptors. Here, we show that Lnk binds to c-Fms. Biological and functional effects of this interaction were examined in macrophages from Lnk-deficient (KO) and WT mice. Clonogenic assays demonstrated an elevated number of M-CFUs in the bone marrow of Lnk KO mice. Furthermore, the M-CSF-induced phosphorylation of Akt in Lnk KO macrophages was increased and prolonged, whereas phosphorylation of Erk was diminished. Zymosan-stimulated production of ROS was increased dramatically in a M-CSF-dependent manner in Lnk KO macrophages. Lastly, Lnk inhibited M-CSF-induced migration of macrophages. In summary, we show that Lnk binds to c-Fms and can blunt M-CSF stimulation. Modulation of levels of Lnk in macrophages may provide a unique therapeutic approach to increase innate host defenses.

Role of Intracellular Tyrosine Residues in Oncogenic KIT- Induced Transformamtion.

Abstract 1435Poster Board I-458Gain-of-function mutations of the receptor tyrosine kinase KIT have been associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myelogenous leukemia (AML). A mutation of aspartic acid to valine (KITD814V) in the activation loop of KIT results in altered substrate recognition and constitutive tyrosine autophosphorylation. However, the intracellular mechanisms that contribute to promiscuous signaling via this mutation are poorly understood. We have previously shown that KITD814V is sufficient to induce ligand independent growth in primary hematopoietic stem and progenitor cells (HSC/P) in vitro as well as transformation in vivo. However, it is not known whether ligand induced (i.e. KITL) signals in vivo contribute to transformation. To assess this, we generated a chimeric receptor (CHR) in which the extracellular domain of KIT was replaced with the human-macrophage colony stimulating factor receptor (h-MCSFR) to inhibit endogenous binding of murine SCF. In vitro thymidine incorporation assay confirmed that the WTCHR is only functionally responsive to human (h)-MCSF stimulation, but not to murine (m)SCF nor murine MCSF stimulation, and that CHRD814V receptor maintains ligand independent growth potential similar to the KITD814V receptor. To determine if the myeloproliferative disease (MPD) could occur in the absence of endogenous ligand binding, a murine transplantation model was utilized to compare CHRD814V and KITD814V-induced transformation. In vivo results demonstrated no significant difference in the onset of MPD in mice bearing KITD814V vs. CHRD814V (median survival 52 days vs. 49 days, n=10 to 20 mice), with similar disease manifestation in the two groups including splenomegaly, hepatomegaly, myeloid cell infiltration and elevated white blood cell counts (n=10 to 20). Transforming potential in the absence of KIT extracellular domain strongly suggested that self-association of KITD814V was sufficient to induce transformation rather than influences from endogenous ligand. To further assess the relative contribution of intracellular tyrosine residues in transformation, a CHRD814V mutant was generated in which seven critical tyrosine residues including the binding sites for Src family kinases, Grb2, p85α regulatory subunit of class IA PI3Kinase, PLC-g, Ras-GAP, and Grb7 were mutated to phenylalanines (i.e.CHRD814V-F7). Our results show that CHRD814V-F7 bearing primary bone marrow cells lost ligand independent growth potential in vitro, suggesting that seven tyrosine induced signaling may play a critical role in KITD814V-induced ligand independent transformation. Consistently, transplantation studies demonstrated that mice bearing bone marrow cells expressing CHRD814V-F7 exhibited a notable delay in MPD development (median survival= 95 days, n=4 to 8). These results suggest that intracellular tyrosines are crucial for ligand independent proliferation and transformation. Next, we individually restored each of the seven tyrosine residues back into CHRD814V-F7 to determine the importance of pathways essential for ligand independent growth and transformation. We found that among the single tyrosine add back receptors, only restoring p85α subunit binding site (Y719) alone was sufficient to induce promiscuous cellular growth, proliferation, and survival. Transplantation studies revealed that restoration of Y719 was sufficient to induce transformation in vivo (median survival= 55 days, n=5). Furthermore, restoration of other individual tyrosine residues demonstrated a notable delay in CHRD814V-induced transformation in vivo (median survival= 83∼120 days, n=5 to 12) and a minimal contribution to ligand independent growth in vitro. Taken together, our results demonstrate: 1) KITD814V induced transformation in vivo occurs in the absence of ligand stimulation; 2) intracellular tyrosine residues play an essential role in KITD814V induced transformation in vitro and in vivo and; 3) of all the critical tyrosine residues present in KIT, presence of tyrosine at position 719 is sufficient to completely restore ligand independent growth in vitro and transformation in vivo. DisclosuresNo relevant conflicts of interest to declare.

Role of Focal Adhesion Kinase in Myelotoxicity, Inflammatory Stress Response and Myeloid Cell Functions.

Abstract 3615Poster Board III-551Focal adhesion kinase (FAK) is a key signaling molecule in focal adhesion signaling and a potential integrator of integrin and growth factor receptor mediated signals. FAK has been implicated in various cellular functions such as growth, survival, migration, adhesion and cytoskeletal reorganization in fibroblasts but its role in hematopoietic stem and progenitors is unknown. To demonstrate the role of FAK in normal and stress-induced hematopoiesis, we generated FAK deficient mice by using a Cre/loxP method from here on termed FAKflox/flox (WT) mice. FAK deletion was induced by injecting poly (I)-poly(C) to FAK flox/flox mice containing the Mx.Cre transgene for one month (FAK-/-). PCR and western blot analysis revealed that after one month of poly (I)-poly(C) induction, hematopoietic cells failed to express detectable levels of FAK in bone marrow (BM), spleen and thymus. To determine the effect of FAK deletion on the development of hematopoietic cells a thorough analysis of the hematopoietic compartment in FAK-/- mice was performed. Total and differential cell counts of peripheral blood revealed significantly high red blood cell distribution width {RDW (%)} and mean cell volume (MCV) in FAK-/- mice compared to WT (n=13, WT; 18.6, 47.2 vs. FAK-/-; 20.06, 48.7, *p<0.05), respectively. In addition, differential basophil counts were significantly less in FAK-/- mice compared to WT (n=13, WT; 0.68 vs. FAK-/-; 0.3 *p<0.04) but all leukocyte populations were present at normal frequencies. Furthermore, platelet counts were significantly higher in FAK-/- peripheral blood compared to WT controls (n=13, WT; 759 vs FAK-/-; 978, *p<0.01). Under basal steady-state conditions, granulopoiesis appeared to be significantly altered in FAK deficient bone marrow (BM), as frequency of granulocytes, but not of other myeloid cells was reduced (n=10, WT; 44.14% vs. FAK-/-; 34.4%, *p<0.0001). Interestingly the frequency of Lin-, c-Kit+, Sca-1+ was also impaired in FAK deficient BM compared to controls (n=9, *p<0.05). FAK deficient BM progenitors displayed significantly lower frequency of colony-forming units compared to WT controls in response to various cytokine combinations (n=6, *p<0.01), which was associated with higher apoptosis in vitro (n=9, *p<0.006). Under conditions of stress, recovery of BM myeloid compartment and Lin−,c-Kit+, Sca-1+ cells following 5-Fluorouracil myeloablation was much slower in FAK-/- mice compared to WT controls (n=3, *p<0.05). Furthermore, the response of myeloid cells to acute inflammatory stress inflicted by intraperitoneal injection of thioglycollate was impaired in FAK-/- mice compared to WT mice (Macrophages: WT; 7.47 × 106 vs. FAK−/−; 3.1 × 106, n=8, *p <0.01. Neutrophils: WT; 5.47 × 106 vs. FAK−/−; 2.1 × 106, n=3, *p <0.05). These results led us to more closely examine the myeloid compartment in these mice. In vitro, FAK-/- macrophage progenitors show reduced growth in response to M-CSF stimulation (n=4, *p <0.01). In addition, deficiency of FAK in macrophages resulted in significant reduction in haptotactic migration in response to M-CSF on extracellular matrix proteins such as fibronectin, laminin and collagen (n=4, *p <0.01). Consistently, a significant reduction in the migration of FAK-/- macrophages was also observed in a wound healing assay which was associated with reduced activation of Rho GTPases including Rac. The reduction in migration of FAK-/- macrophages was associated with a significant decrease in adhesion on fibronectin, laminin and collagen. The impaired migration and adhesion of FAK-/- macrophages was observed in spite of comparable levels of F4/80 as well as integrin (α4β1 & α5β1) expression. Consistent with enhanced neutrophil apoptosis and reduced frequency under basal conditions, FAK deficient BM derived neutrophil progenitors (BMNs) show reduced growth and cycling in response to G-CSF stimulation (n=4, *p <0.01). Deletion of FAK in BMNs led to increased apoptosis upon cytokine withdrawal, which was associated with reduced activation of AKT and increased caspase-3 cleavage compared to controls. Taken together, our findings indicate that FAK plays a vital role in modulating physiological stress response to myeloablation, inflammation as well as in regulating several functions in macrophages and neutrophils. Disclosures:No relevant conflicts of interest to declare.

SH3BP2 is a Critical Regulator of Macrophage and Osteoclast Response to M-CSF and RANKL Stimulation

The protein Src homology 3 domain-binding protein 2 (SH3BP2) is a regulator of tumor necrosis factor (TNF)-alpha generation in macrophages and of osteoclast differentiation. SH3BP2 regulates bone marrow monocyte responses to macrophage and osteoclast differentiation signals downstream of the receptors for macrophage colony-stimulating factor and receptor activator of nuclear factor kappaB ligand. SH3BP2 is a potential target for the development of novel anti-TNF-alpha therapeutic interventions as well as a target for suppression of osteoclastogenesis.