Presence Of M-CSF Research Articles

Proinflammatory and proosteoclastogenic potential of peripheral blood mononuclear cells from Gaucher patients: Implication for bone pathology.

Gaucher disease (GD) is caused by mutations in the GBA gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to the accumulation of the glycolipid glucocerebroside in the lysosomes of cells of monocyte/macrophage system. Bone compromise in Gaucher disease patients is the most disabling aspect of the disease. However, pathophysiological aspects of skeletal alterations are still poorly understood. On the other hand it is well known that inflammation is a key player in GD pathology. In this work, we revealed increased levels of the proinflammatory CD14(+)CD16(+) monocyte subset and increased inflammatory cytokine production by monocytes and T cells in the circulation of GD patients. We showed increased levels of osteoclast precursors in PBMC from patients and a higher expression of RANKL in the surface of T cells. PBMC from patients presented higher osteoclast differentiation compared to healthy controls when cultured in the presence of M-CSF alone or in combination with RANKL. In vitro treatment with Velaglucerase reduced osteoclast levels to control levels. On the other hand THP-1 derived osteoclast precursors cultured in the presence of conditioned media from PBMC of GD patients presented higher differentiation to active osteoclasts. This induction involved TNF-α and RANKL.

MafB antagonizes phenotypic alteration induced by GM-CSF in microglia

Microglia are tissue-resident macrophages which are distributed throughout the central nervous system (CNS). Recent studies suggest that microglia are a unique myeloid population distinct from peripheral macrophages in terms of origin and gene expression signature. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a pleiotropic cytokine regulating myeloid development, has been shown to stimulate proliferation and alter phenotype of microglia in vitro. However, how its signaling is modulated in microglia is poorly characterized. MafB, a bZip transcriptional factor, is highly expressed in monocyte-macrophage lineage cells including microglia, although its role in microglia is largely unknown. We investigated the crosstalk between GM-CSF signaling and MafB by analyzing primary microglia. We found that Mafb-deficient microglia grew more rapidly than wild-type microglia in response to GM-CSF. Moreover, the expression of genes associated with microglial differentiation was more downregulated in Mafb-deficient microglia cultured with GM-CSF. Notably, such differences between the genotypes were not observed in the presence of M-CSF. In addition, we found that Mafb-deficient microglia cultured with GM-CSF barely extended their membrane protrusions, probably due to abnormal activation of RhoA, a key regulator of cytoskeletal remodeling. Altogether, our study reveals that MafB is a negative regulator of GM-CSF signaling in microglia. These findings could provide new insight into the modulation of cytokine signaling by transcription factors in microglia.

Glycolysis regulates macrophage differentiation into IL-10 producing phenotype (INM6P.342)

Abstract There are a lot of reports indicating that cellular metabolic state or some metabolites regulate the phenotype of immune cells. In case of macrophages (Mφ), it is believed that regulatory and inflammatory Mφ shifts ATP production to each oxidative phosphorylation and glycolysis, respectively. However, it is not clear how metabolic pathway affects Mφ in the acquisition of their specific phenotypes. In this report, we tried to reveal how glycolysis affects Mφ during the acquisition of IL-10 producing phenotype. We used in vitro differentiated human monocyte derived Mφ cultured in the presence of M-CSF or M-CSF plus IFNγ as IL-10 producing-Mφ (M-Mφ) or inflammatory Mφ (Mγ-Mφ), respectively. M-Mφ showed regulatory Mφ-like phenotypes and produced more IL-10 when it was stimulated with LPS compared to Mγ-Mφ. Interestingly, M-Mφ differentiated with glycolysis inhibitors (2-deoxy-D-glucose or DCA) produced significantly lower amount of IL-10 and lager amount of IL-6 compared to M-Mφ differentiated without glycolysis inhibitors. Glycolysis inhibited M-Mφ reduced the expression of regulatory Mφ marker genes (MRC1 and CCL22) and exhibited distinct pattern of cell surface antigens (CD163, CD206, CD209 and HLA-DR) compared to M-Mφ. DNA microarray analysis revealed that inhibition of glycolysis affects gene expression profiles during Mφ differentiation. Our finding suggests that glycolysis plays key roles in the course of differentiation of Mφ into IL-10 producing phenotype.

IL-34- and M-CSF-induced macrophages switch memory T cells into Th17 cells via membrane IL-1α.

Macrophages orchestrate the immune response via the polarization of CD4(+) T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M-CSF and IL-34 induce the differentiation of monocytes into IL-10(high) IL-12(low) immunoregulatory macrophages, which are similar to tumor-associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M-CSF (M-CSF macrophages) or IL-34 (IL-34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4(+) T cells. Taken together, our results show that M-CSF-, IL-34 macrophages, and TAMs switch non-Th17 committed memory CD4(+) T cells into conventional CCR4(+) CCR6(+) CD161(+) Th17 cells, expressing or not IFN-gamma. Contrary, the pro-inflammatory GM-CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL-1α (mIL-1α), which is constitutively expressed by M-CSF-, IL-34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.

Development of a Novel Phthalimide Derivative, Preclinical Effects on High-Risk Myeloma Cells and Osteoclasts

BACKGROUND: Despite recent advances in the use of newly developed drugs including immune-modulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide and proteasome inhibitors such as bortezomib, carfilzomib, and MLN9708, MM is still an incurable disease. In particular, MM patients harboring 17p deletion, t(14;16), t(14;20), or t(4;14) are classified as a high-risk group and have shown significantly shorter survival. With the goal of helping prolong the survival of these high-risk MM patients, we screened 29 synthetic phthalimide derivatives and found a novel compound, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11), which induced the apoptosis of KMS34 cells with t(4;14) and del17p13.PURPOSE:The purpose of this project is to clarify preclinical effects of the synthetic phthalimide derivative, TC11, on high-risk MM cell lines and osteoclasts. Namely, anti-myeloma and anti-osteoclastogenic activities and pharmacokinetic study in mice were shown. We also try to isolate directly binding molecules. Safety issues including hematological toxicities and teratogenicity were also discussed.METHODS AND RESULTS: TC11 significantly inhibited growth of MM cell lines (IC50 4-8μM) including KMS34 and KMS11 cells which have high-risk chromosomal abnormalities. TC11 also suppressed the proliferation of all of the bone marrow cells obtained from the MM patients, in a dose-dependent manner. TC11 increased annexin V-positive fraction and induced apoptosis. TC11 was injected intraperitonealy into myeloma (KMS34 and KMS11 cells)-bearing lcr/SCID mice, and anti-myeloma activity was evaluated in vivo. Twenty mg/kg of TC11 significantly inhibited growth of KMS34 or KMS11-derived plasmacytomas. Apoptosis of MM cells was observed by histopathological examination. In order to evaluate hematological toxicity of TC11, growth of colony-forming cells was examined. In the presence of 5μM of TC11, formation of CFCs was not significantly suppressed, suggesting low hematopoietic toxicity. In the pharmacokinetic analyses using lcr mice, the plasma concentrations of TC11 was examined; Cmaxwas 18.1μM at 1.5hr (Tmax), and T1/2 was 2.5hr, when 100mg/kg of TC11 was injected. If 20mg/kg was injected, Cmaxwas 2.1μM at 1.0hr (Tmax), and T1/2 was 1.2hr. Oral administration of TC11 to Icr mice was safely carried out, and results of pharmacokinetic study will be shown. Aiming at the therapeutic use of TC11 to bone disease, anti-osteoclastogenic activity was examined. Mouse bone marrow mononuclear cells were incubated in the presence of M-CSF and RANK-ligand. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in number in the presence of 1μM of TC11. It was also found that 1μM of TC11 inhibited bone resorption by pit assay. We have identified nucleophosmin 1 (NPM1) and α-tubulin as TC11-binding molecules using our unique in vitro selection system using mRNA display, in vitro virus (IVV) method. However, cereblon (CRBN) was not detected as a TC11-binding protein by this method. The immunofluorescent analysis showed that TC11-treated cells exhibited elevated levels of α-tubulin fragmentation. Together with our previous observation of induction of centrosomal disruption of HeLa cells by NPM1-knock down, TC11 may cause anti-myeloma effects via mitotic catastrophe.CONCLUSION: We have demonstrated that TC11, a novel phthalimide derivative, has anti-tumor activity against MM cells with high-risk genetic abnormality including del 17p and t(4;14), in vitro and in vivo. This novel compound also down-regulates the differentiation and function of osteoclasts. Our data provide a strong preclinical rationale for TC11 as a safe and effective drug for the treatment of high-risk MM patients with bone disease. The actions of this drug relating to α-tubulin and NPM1 remain to be further investigated. TC11 exerts its anti-myeloma effect via molecular interactions which do not involve CRBN. In addition, TC11 does not form racemate and is expected to lack teratogenicity. The results of our present study suggest that new phthalimide derivatives other than thalidomide, lenalidomide and pomalidomide could be developed by drug designing for the treatment of MM. DisclosuresNo relevant conflicts of interest to declare.

Therapy of peritoneally disseminated colon cancer by TAP-deficient embryonic stem cell-derived macrophages in allogeneic recipients.

We established a method to generate a large quantity of myeloid lineage cells from mouse embryonic stem (ES) cells, termed ES cell-derived proliferating myeloid cell lines (ES-ML). ES-ML continuously proliferated in the presence of M-CSF and GM-CSF. ES-ML genetically modified to express an anti-HER2 (neu) mAb single-chain V region fragment reduced the number of cocultured mouse Colon-26 cancer cells expressing HER2. Stimulation of ES-ML with IFN-γ plus LPS or TNF resulted in almost complete killing of the Colon-26 cells by the ES-ML, and the cytotoxicity was mediated, in part, by NO produced by ES-ML. When ES-ML were injected into mice with i.p. established Colon-26 tumors, they efficiently infiltrated the tumor tissues. Injection of ES-ML with rIFN-γ and LPS inhibited cancer progression in the mouse peritoneal cavity. Coinjection of TNF-transfected or untransfected ES-ML with rIFN-γ inhibited cancer growth and resulted in prolonged survival of the treated mice. In this experiment, transporter associated with Ag processing (TAP)1-deficient ES-ML exhibited therapeutic activity in MHC-mismatched allogeneic recipient mice. Despite the proliferative capacity of ES-ML, malignancy never developed from the transferred ES-ML in the recipient mice. In summary, TAP-deficient ES-ML with anticancer properties exhibited a therapeutic effect in allogeneic recipients, suggesting the possible use of TAP-deficient human-induced pluripotent stem cell-derived proliferating myeloid cell lines in cancer therapy.

Generation of BTNL8+ Inducible Tregs By Allogeneic Human Regulatory Macrophages Is IDO- and B7-Dependent.

Purpose: This study identifies the phenotypic and functional changes that human T cells undergo when exposed to regulatory macrophages. Methods: Human regulatory macrophages (M regs) represent a unique and stable state of macrophage activation, characterized by their ability to suppress activated T cells. M regs arise from CD14+ monocytes over a 6-day culture period in the presence of M-CSF, human serum, and a final 24-hour pulse of IFN-γ. In preclinical models, pre-transplant administration of donor-derived M regs prolonged allograft survival. This graft-protective effect initially depended upon metabolically-competent cells, but persisted beyond the lifespan of the M regs, implying regulation of recipient T cells by M regs. Here, mechanisms by which human M regs influence allogeneic T cell responses in vitro are examined. Results: Phenotypic and functional changes imposed by human M regs on allogeneic T cells were investigated by flow cytometry, whole-genome gene expression profiling, and secondary suppression assays. After exposure to M regs, CD4+ T cells were induced to express FoxP3, CD25, CTLA4, OX40, GITR, ICOS, EBI3, and BTNL8. Such FoxP3+ T cells generated in M reg cocultures suppressed polyclonal T cell proliferation and inhibited TNFα-induced dendritic cell maturation. The Treg-specific demethylated region (TSDR) of the FOXP3 gene in M reg-cocultured CD4+ T cells was 5% demethylated. The M reg-induced regulatory T cell (Treg) phenotype was not alloantigen-dependent, but was contact-dependent, and could be abolished by B7 blockade with CTLA4-Ig or by indoleamine 2,3-dioxygenase (IDO) inhibition with 1 mM 1-L-methyltryptophan. Tacrolimus and rapamycin did not affect FoxP3+ T cell development. Conclusions: M regs drive an antigen non-specific, B7- and contact-dependent induction of activated induced Tregs through a mechanism partially dependent on IDO. This finding supports the concept that pre-transplant treatment of recipients with donor-derived M regs engenders a pro-tolerogenic state and hints at a feedforward mechanism by which it could be perpetuated. Clinical applications of M reg immunosuppressive therapy are currently being pursued within The ONE Study framework.

THU0499 Hm71224, A Novel Oral BTK Inhibitor, Inhibits Human Immune Cell Activation: New Drug Candidate to Treat B-Cell Associated Autoimmune Diseases

BackgroundBruton tyrosine kinase (BTK), a Tec family kinase, is involved in B cell activation and osteoclast formation. As BTK plays a central role in B cell receptor (BCR) signaling and...

House dust mite increases IL-33 and thymic stromal lymphopoietin (TSLP) gene expression in response to human rhinovirus 1B in murine bone marrow-derived macrophages. (HYP6P.276)

Abstract Acute viral infections are the most frequent cause of asthma exacerbations, and are therefore responsible for the majority of healthcare costs attributed to asthma. Human rhinovirus is the most common identified one in children and adults. It has been shown that IL-33- driven mechanism initiates TH2 responses to the clinically relevant allergens house dust mite (HDM). We have shown that TSLP is increased in asthma exacerbation. Alveolar macrophages have higher expression of IL-33 and TSLP in asthma. To determine whether HDM can have an effect on IL-33 and TSLP gene expression in response to human rhinovirus (RV), we treated bone marrow-derived macrophages (BMM) with HDM in the presence of M-CSF and infected them with RV1B. RV1B induced expression of IL-33 (2-fold, P-value<0.001) and TSLP (2-fold P-value<0.05) in HDM- treated macrophages after 24 hours compared to HDM non-treated BMM. RV1B infection caused an increase in IL-33 in IL-33 (P-value<0.01) and TSLP ( P-value<0.01) in HDM treated BMM compared to UV-treated RV1B group. There was no increase in IL-33 and TSLP gene expression in response to RV1B in the HDM-treated BMM at eight hours compared to UV-treated RV1B group, while HDM non-treated BMM showed increase in IL-33 (P-value<0.01) and TSLP (P-value=0.059 ) in response to RV1B compared to UV-treated RV1B. These results suggest that HDM increases IL-33 and TSLP gene expression in macrophages in response to RV1B which can play a role in asthma exacerbations.

The role of endothelin receptors (ETRA/B) in fibrocyte differentiation

Introduction Scleroderma (SSc) is an autoimmune connective tissue disease of unknown aetiology. Pulmonary involvement including the development of pulmonary arterial hypertension (PAH) is characterised by vascular remodelling, collagen deposition and expression of connective tissue growth factor (CTGF). CD14+ monocytes can differentiate into spindle shaped cells termed ‘fibrocytes’. Fibrocytes express haematopoietic and mesenchymal markers including collagen, and amplify inflammatory/immune responses via antigen presentation and chemokine secretion. Fibrocyte differentiation is enhanced by fibrogenic cytokines including PDGF. The role fibrocytes play in promoting PAH in SSc is unknown. Methods CD14+ PBMCs were isolated from SSc and healthy donor blood. Fibrocyte differentiation in the presence of MCSF and/or ET-1 was assessed after 14 days. The effect of endothelin receptor (ETR) antagonists (selective/dual) on fibrocyte differentiation (n = 6) was investigated. SSc and control fibrocyte secretomes were assessed by ELISA (n = 6), and the effects on fibroblast-mediated gel contraction determined. Results MCSF and ET-1 alone and in combination induced fibrocyte differentiation (P < 0.05). SSc fibrocytes exhibited enhanced differentiation from CD14+ PBMCs than healthy control donors in response to MCSF (P < 0.05), ET-1 (P < 0.05) and in combination (P < 0.01). ETR antagonists BQ123 (ETRA), BQ788 (ETRB) and Bosentan (ETRA/B) inhibited MCSF induced fibrocyte differentiation. CTGF secretion was elevated in SSc compared to control fibrocytes (P < 0.05) cultured with MCSF. Conditioned media from SSc fibrocytes promoted gel contraction by control pulmonary fibroblasts (P < 0.05). Discussion CD14+ SSc PBMCs readily differentiate into fibrocytes in response to ET-1 and MCSF via ETRA and ETRB. Our data suggests fibrocytes contribute to the development of PAH in SSc via a paracrine mechanism modulating the functional activities of resident tissue fibroblasts.

Bone Morphogenetic Proteins Signal Via SMAD and Mitogen-activated Protein (MAP) Kinase Pathways at Distinct Times during Osteoclastogenesis

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII(fl/fl);lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.

Macrophages Engulf Tumor B Cells Coated With Rituximab More Effectively Under The Hypoxic Condition

Growing evidence has been accumulated indicating that the tumor microenvironment plays a pivotal role in the pathogenesis of various tumors. Macrophages are one of the most important players in such a microenvironment, and those residing in tumor tissues are called “Tumor-Associated Macrophages (TAM)”. The TAM content predicts survival in follicular lymphoma patients following treatment, and a high TAM count was associated with a poor clinical course after conventional chemotherapy before the rituximab era. However, immunochemotherapy with rituximab is able to circumvent the unfavorable outcome associated with a high TAM count. This finding thus favors the hypothesis that TAM may act synergistically with anti-CD20–targeted therapy. Hypoxia is a key feature of the microenvironment of cancer cells, and actively participates in tumor progression. In this study, we investigated the impact of hypoxia on the interaction between rituximab and TAM. Macrophages were prepared by culturing isolated monocytes from adult volunteers at the Hokkaido Blood Bank in the presence of M-CSF for seven days in a humidified incubator containing 20% O2, 5% CO2 and 75% N2 (normoxic macrophages) or 1% O2, 5% CO2 and 94% N2(hypoxic macrophages). We used CD20-positive B cells from patients with chronic lymphocytic leukemia (CLL B cells) as target cells. When CLL B cells were opsonized with rituximab, the cell-binding assay showed that the hypoxic macrophages bound more effectively than normoxic macrophages (29.2±0.6% and 18.5±0.9%, respectively, p&lt;0.01). We then performed phagocytosis assays using standard cytospin techniques and microscopic evaluation of stained phagocytic cells. Again, the percentage of phagocytosis of CLL B cells in the presence of rituximab was significantly higher under hypoxic conditions compared to those cultured under normoxic conditions (22.5±1.3% and 13.9±0.9%, respectively, p&lt;0.01). We next investigated the expression of Fc receptors on macrophages cultured in the different oxygen conditions. A flow cytometric analysis revealed that the expression levels of CD16, but not CD32 or CD64, were enhanced on hypoxic macrophages compared to normoxic macrophages, and this upregulation of CD16 was considered to contribute to the increased binding and phagocytosis of macrophages under hypoxic conditions. The phenotype and functional activities of macrophages can be modulated by a number of cytokines and microbial products. Among these cytokines, IL-10 was reported to be the best inducer of macrophages with high phagocytic activity. When IL-10 was added in the culture 24 hours before the phagocytosis assay, the phagocyte rate of hypoxic, but not normoxic, macrophages was significantly enhanced. Intriguingly, the expression levels of CD16 on hypoxic macrophages were upregulated by IL-10 treatment, and this upregulation was not seen in the normoxic macrophages. Taken together, these findings indicate that the hypoxic microenvironment facilitated the rituximab-mediated binding and phagocytic activity of TAM through the upregulation of CD16 expression. IL-10 could therefore be a possible effector modulator that enhances the activity of rituximab in the hypoxic microenvironment. Disclosures: No relevant conflicts of interest to declare.

S139 The role of endothelin receptors (ETRA/B) in fibrocyte differentiation

IntroductionScleroderma (SSc) is an autoimmune connective tissue disease of unknown aetiology. Pulmonary involvement including the development of pulmonary arterial hypertension (PAH) is characterised by vascular remodelling, collagen deposition and expression...

Abstract 638: High Salt (NaCl) Affects H3K4 Trimethylation And Alternative Macrophage Activation

High dietary salt (NaCl) is a major risk factor for cardiovascular disease (CVD). We have shown that macrophages play a key role in the regulation of interstitial tonicity and that NaCl-induced hypertonicity amplifies T cell (Th17) polarization. We therefore hypothesize that NaCl-induced hypertonicity also affects macrophage polarization. Here we have investigated the effect of NaCl-induced hypertonicity on the IL4/IL13-induced alternative activation of M2 macrophages (AAM). Bone marrow cells from C57BL/6 mice were differentiated in the presence of M-CSF for 7 days to generate M0 macrophages. To induce AAM, M0 macrophages were then incubated with IL4/IL13 for 24h in the presence of 0-40mM NaCl. Quantitative RT-PCR was used to analyze AAM signature genes (e.g. Mrc1, Arg1, Fizz1, Ym-1, PD-L2). NaCl specifically blunted the upregulation of these genes and Klf4 and Irf4 (two transcription factors essential for AAM polarization). In contrast, osmotic stress by mannitol or urea did not have an effect. In previous experiments Sgk1 played a pivotal role in Th17 polarization. In contrast, Sgk1 was not involved in the NaCl-effect on AAM macrophages. It has been shown that histone modifications are essential for AAM. In RAW264.3 macrophages, IL4/IL13 induced an increase in H3K4 trimethylation of a number of genes, including Mrc1. However this H3K4 trimethylation was blunted by NaCl. We also found that incubation with increased concentrations of NaCl altered STAT6 phosphorylation upon IL4/IL13 stimulation. We propose that NaCl-induced hypertonicity alters chromatin modifications and signaling cascades important for the expression of genes essential for AAM polarization and function.

Pre-Weaning Growth Hormone Treatment Ameliorates Bone Marrow Macrophage Inflammation in Adult Male Rat Offspring following Maternal Undernutrition

Maternal undernutrition (UN) is associated with the development of obesity and metabolic complications in adult offspring. While the role of inflammation in obesity and related comorbidities has been well established, there is little evidence regarding the effects of maternal UN-induced programming on immune function in male adult offspring. This study examines the effects growth hormone (GH), which is known to induce anti-inflammatory effects, on maternal UN-induced bone marrow macrophage (BMM) function in adult male offspring. Sprague-Dawley rats were assigned to chow (C) or UN (50% ad libitum; UN) diet throughout gestation. Male C and UN pups received saline (CS/UNS) or GH (2.5 µg/g/d; CGH/UNGH) from day 3–21. Bone marrow hematopoietic cells were differentiated to a macrophage phenotype in the presence of M-CSF (50 ng/ml). Differentiated bone marrow macrophages (BMM) were stimulated with LPS (100 ng/ml) for 6 h. UNS-derived BMM had significantly increased secretion and expression of IL-1β and IL-6 following LPS stimulation. This was accompanied by increased expression of IL-1R1, IL-6R and TLR4. Pre-weaning GH treatment reversed this pro-inflammatory phenotype. Furthermore UNGH displayed increased expression of markers of alternative (M2) macrophage activation, mannose receptor and PPARγ. This study demonstrates that fetal UN exposure primes hematopoietic immune cells to a more potent pro-inflammatory phenotype with heightened cytokine secretion and receptor expression. Furthermore these cells are pre-disposed to pro-inflammatory M1 macrophage phenotype which has wide-reaching and important effects in terms of obesity and metabolic disease.

Generation of a large number of functional dendritic cells from human monocytes expanded by forced expression of cMYC plus BMI1

Anticancer vaccination therapies with monocyte-derived dendritic cells (DC) are widely conducted. A large number of primary monocytes (approximately 10(8) cells) are needed to generate the number of DC required to achieve an effect upon vaccination, and monocytes are usually purified from peripheral blood mononuclear cells obtained by apheresis procedure, which is somehow invasive for cancer patients. As a means to facilitate the generation of DC for therapeutic use, we herein report a method to amplify human monocytes. We found that lentivirus-mediated transduction of cMYC along with BMI1 induced proliferation of CD14(+) monocytes derived from 9 out of 12 blood donors, and we named the monocyte-derived proliferating cells CD14-ML. Their proliferation continued for 3-5 weeks in the presence of M-CSF and GM-CSF, resulting in 20-1000-fold amplification. Importantly, the expanded CD14-ML differentiated into fully functional DC (CD14-ML-DC) upon the addition of IL-4 to the culture. We successfully stimulated autologous CD8(+) T cells with CD14-ML-DC pulsed with cytomegalovirus peptide or MART-1 peptide to generate antigen-specific CTL lines. This is the first report describing the method for in vitro expansion of human peripheral blood monocytes.

DAP12 Overexpression Induces Osteopenia and Impaired Early Hematopoiesis

ITAM-bearing transmembrane signaling adaptors such as DAP12 and FcRγ are important players in bone homeostasis, but their precise role and functions are still unknown. It has been shown that osteoclast differentiation results from the integration of the RANK and of the DAP12 and FcRγ signaling pathways. DAP12-deficient mice suffer from a mild osteopetrosis and culture of their bone marrow cells in the presence of M-CSF and RANKL, fails to give rise to multinucleated osteoclasts. Here, we report that mice overexpressing human DAP12 have an osteopenic bone phenotype due to an increased number of osteoclasts on the surface of trabecular and cortical bone. This enhanced number of osteoclasts is associated with an increased number of proliferating myeloid progenitors in Tg-hDAP12 mice. It is concomitant with an arrest of B cell development at the Pre-Pro B/Pre B stage in the bone marrow of Tg-hDAP12 mice and important decrease of follicular and marginal B cells in the spleen of these animals. Our data show that the overexpression of DAP12 results in both increased osteoclastogenesis and impaired hematopoiesis underlining the relationship between bone homeostasis and hematopoiesis.

Restriction of HIV-1 Replication in Primary Macrophages by IL-12 and IL-18 through the Upregulation of SAMHD1

Monocyte-derived macrophages (MDM) can polarize into different subsets depending on the environment and the activation signal to which they are submitted. Differentiation into macrophages allows HIV-1 strains to infect cells of the monocytic lineage. In this study, we show that culture of monocytes with a combination of IL-12 and IL-18 led to macrophage differentiation that was resistant to HIV-1 infection. In contrast, M-CSF-derived MDM were readily infected by HIV-1. When monocytes were differentiated in the presence of M-CSF and then further treated with IL-12/IL-18, cells became resistant to infection. The restriction on HIV-1 replication was not dependent on virus entry or coreceptor expression, as vesicular stomatitis virus-pseudotyped HIV-1 replication was also blocked by IL-12/IL-18. The HIV-1 restriction factor sterile α motif and HD domain-containing protein-1 (SAMHD1) was significantly overexpressed in IL-12/IL-18 MDM compared with M-CSF MDM, and degradation of SAMHD1 by RNA interference or viral-like particles carrying the lentiviral protein Vpx restored HIV-1 infectivity of IL-12/IL-18 MDM. SAMHD1 overexpression induced by IL-12/IL-18 was not dependent on IFN-γ. Thus, we conclude that IL-12 and IL-18 may contribute to the response against HIV-1 infection through the induction of restriction factors such as SAMHD1.

The Response of Human Macrophages to β-Glucans Depends on the Inflammatory Milieu

Backgroundβ-glucans are fungal cell wall components that bind to the C-type lectin-like receptor dectin-1. Polymorphisms of dectin-1 gene are associated with susceptibility to invasive fungal infection and medically refractory ulcerative colitis. The purpose of this study has been addressing the response of human macrophages to β-glucans under different conditions mimicking the composition of the inflammatory milieu in view of the wide plasticity and large range of phenotypical changes showed by these cells, and the relevant role of dectin-1 in several pathophysiological conditions.Principal FindingsSerum-differentiated macrophages stimulated with β-glucans showed a low production of TNFα and IL-1β, a high production of IL-6 and IL-23, and a delayed induction of cyclooxygenase-2 and PGE2 biosynthesis that resembled the responses elicited by crystals and those produced when phagosomal degradation of the phagocytic cargo increases ligand access to intracellular pattern recognition receptors. Priming with a low concentration of LPS produced a rapid induction of cyclooxygenase-2 and a synergistic release of PGE2. When the differentiation of the macrophages was carried out in the presence of M-CSF, an increased expression of dectin-1 B isoform was observed. In addition, this treatment made the cells capable to release arachidonic acid in response to β-glucan.ConclusionsThese results indicate that the macrophage response to fungal β-glucans is strongly influenced by cytokines and microbial-derived factors that are usual components of the inflammatory milieu. These responses can be sorted into three main patterns i) an elementary response dependent on phagosomal processing of pathogen-associated molecular patterns and/or receptor-independent, direct membrane binding linked to the immunoreceptor tyrosine-based activation motif-bearing transmembrane adaptor DNAX-activating protein 12, ii) a response primed by TLR4-dependent signals, and iii) a response dependent on M-CSF and dectin-1 B isoform expression that mainly signals through the dectin-1 B/spleen tyrosine kinase/cytosolic phospholipase A2 route.

Molecular differences of NLRP3 inflammasome activation in LPS-activated human monocyte-derived macrophage subtypes

Event Abstract Back to Event Molecular differences of NLRP3 inflammasome activation in LPS-activated human monocyte-derived macrophage subtypes Marietta M. Budai1, Judit Danis1, Ágnes Becsei1, Aliz Varga1, László Csernoch1, József Tőzsér2 and Szilvia Benkő1* 1 University of Debrecen, Medical and Health Science Center, Faculty of Medicine, Department of Physiology, Hungary 2 University of Debrecen, , Medical and Health Science Center, Faculty of Medicine, Department of Biochemistry and Molecular Biology, Department of Biochemistry and Molecular Biology, Hungary IL-1beta is a “master” cytokine that has indispensable roles in orchestrating effective innate and adaptive immune responses. It is produced in an inactive precursor form that is cleaved to active cytokine by protein complexes called Nlrp3 inflammasomes (1). While our knowledge on the general mechanisms involved in Nlrp3 inflammasome function and on its regulation is rapidly increasing, it is also getting clear that the actual outcome of the activation (like IL-1beta production) strongly depends on the cell type and on the presence or absence of various intracellular or extracellular modulators (2). Depending on their localization macrophages can develop into a wide range of phenotypes. Macrophages differentiated in the presence of GM-CSF (GM-MFs) develop inflammatory phenotype, while cells differentiated in the presence of M-CSF (M-MFs) possess anti-inflammatory characteristics and function in wound healing and tissue repair. Our results show that following LPS treatment GM-MFs produce high IL-1beta, while M-MFs produce low IL-1beta with a different time- and concentration kinetics. We found significant differences in basal and LPS-induced expression of Nlrp3, procaspase-1 and ASC between the two MF types. We found that LPS-treated GM-MFs are able to release ATP and produce IL-1beta, while M-MFs require ATP supplementation for IL-1beta secretion. We have also found expression differences in the proteins responsible for ATP release, recognition and degradation as well as in the activation of key signal transduction pathways. Furthermore, we will show that different nucleosides have strong and differential regulatory effects of NLRP3 inflammasome function. Acknowledgements This work was supported by TÁMOP 4.2.2.A-11/1/KONV-2012-0023 "VÉD-ELEM" grant and UD Faculty of Medicine Research Fund - Bridging Fund.