Macrophage Colony-stimulating Factor Neutralizing Research Articles

Bioactive materials from berberine-treated human bone marrow mesenchymal stem cells promote alveolar bone regeneration by regulating macrophage polarization.

Alveolar bone regeneration has been strongly linked to macrophage polarization. M1 macrophages aggravate alveolar bone loss, whereas M2 macrophages reverse this process. Berberine (BBR), a natural alkaloid isolated and refined from Chinese medicinal plants, has shown therapeutic effects in treating metabolic disorders. In this study, we first discovered that culture supernatant (CS) collected from BBR-treated human bone marrow mesenchymal stem cells (HBMSCs) ameliorated periodontal alveolar bone loss. CS from the BBR-treated HBMSCs contained bioactive materials that suppressed the M1 polarization and induced the M2 polarization of macrophages in vivo and in vitro. To clarify the underlying mechanism, the bioactive materials were applied to different animal models. We discovered macrophage colony-stimulating factor (M-CSF), which regulates macrophage polarization and promotes bone formation, a key macromolecule in the CS. Injection of pure M-CSF attenuated experimental periodontal alveolar bone loss in rats. Colony-stimulating factor 1 receptor (CSF1R) inhibitor or anti-human M-CSF (M-CSF neutralizing antibody, Nab) abolished the therapeutic effects of the CS of BBR-treated HBMSCs. Moreover, AKT phosphorylation in macrophages was activated by the CS, and the AKT activator reversed the negative effect of the CSF1R inhibitor or Nab. These results suggest that the CS of BBR-treated HBMSCs modulates macrophage polarization via the M-CSF/AKT axis. Further studies also showed that CS of BBR-treated HBMSCs accelerated bone formation and M2 polarization in rat teeth extraction sockets. Overall, our findings established an essential role of BBR-treated HBMSCs CS and this might be the first report to show that the products of BBR-treated HBMSCs have active effects on alveolar bone regeneration.

Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages.

Tissue macrophages are important components of tissue homeostasis and inflammatory pathologies. In the peritoneal cavity, resident macrophages interact with a variety of immune cells and can exhibit broad range of phenotypes and functions. Forkhead-box-P3 (FOXP3)+ regulatory T cells (Tregs) play an indispensable role in maintaining immunological tolerance, yet whether, and how the pathological condition that results from the lack of functional Tregs affects peritoneal macrophages (PM) is largely unknown. We used FOXP3-deficient scurfy (Sf) mice to investigate PM behavior in terms of the missing crosstalk with Tregs. Here, we report that Treg deficiency induced a marked increase in PM numbers, which was reversed after adoptive transfer of CD4+ T cells or neutralization of macrophage colony-stimulating factor. Ex vivo assays demonstrated a pro-inflammatory state of PM from Sf mice and signs of excessive activation and exhaustion. In-depth immunophenotyping of Sf PM using single-cell chipcytometry and transcriptome analysis revealed upregulation of molecules involved in the initiation of innate and adaptive immune responses. Moreover, upon transfer to non-inflammatory environment or after injection of CD4+ T cells, PM from Sf mice reprogramed their functional phenotype, indicating remarkable plasticity. Interestingly, frequencies, and immune polarization of large and small PM subsets were dramatically changed in the FOXP3-deficient mice, suggesting distinct origin and specialized function of these subsets in inflammatory conditions. Our findings demonstrate the significant impact of Tregs in shaping PM identity and dynamics. A better understanding of PM function in the Sf mouse model may have clinical implication for the treatment of immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, and other forms of immune-mediated enteropathies.

Bone marrow mesenchymal stem cells tune the differentiation of myeloid-derived suppressor cells in bleomycin-induced lung injury

BackgroundBone marrow mesenchymal stem cells (BMSC) transfer has been attempted as a therapeutic strategy in experimental lung injury and fibrosis. Reduction of neutrophilic infiltration is one of the mechanisms involved in this effect. However, the mechanisms by which BMSC modulate neutrophil remains unknown.Methods and resultsExposure of mice to bleomycin (BLM) resulted in significant accumulation of cells that express neutrophilic markers Gr-1HighCD11b+Ly-6GHighF4/80―CD115―CD49d―. These cells lacked immunosuppressive activity and could not be defined as myeloid-derived suppressor cells (MDSC). When BMSC were administrated to BLM-treated mice, they tuned the differentiation of Gr-1HighCD11b+ toward Gr-1LowCD11b+ cells. Gr-1LowCD11b+ cells exhibited unsegmented nuclei and expressed F4/80, Ly-6C, CD49d, and CD115 markers. These cells had potent immunosuppressive activity and thus could be defined as monocytic MDSC. As a result of such immunoregulation, BMSC mediated a decrease of pro-inflammatory products and amelioration of lung injury in BLM-treated mice. Further study using antibody array showed increased expression of macrophage colony-stimulating factor (M-CSF) in BMSC-treated mice. Accumulation of Gr-1LowCD11b+ cells in BMSC-treated mice was abrogated in M-CSF neutralizing mice. The beneficial effect of BMSC was independent of the ability of the cells to engraft in lung and in vitro coculture study of BMSC with Gr-1+CD11b+ cells showed that the induction of Gr-1LowCD11b+ cells by BMSC was independent of cell-cell contact.ConclusionsThese results document the generation of Gr-1HighCD11b+ cells in BLM-treated mice, and suggest that BMSC tune the differentiation of Gr-1HighCD11b+ toward Gr-1LowCD11b+ cells and therefore inhibit the progression of BLM-induced lung injury.

Neurons and astroglia govern microglial endotoxin tolerance through macrophage colony-stimulating factor receptor-mediated ERK1/2 signals

Endotoxin tolerance (ET) is a reduced responsiveness of innate immune cells like macrophages/monocytes to an endotoxin challenge following a previous encounter with the endotoxin. Although ET in peripheral systems has been well studied, little is known about ET in the brain. The present study showed that brain immune cells, microglia, being different from peripheral macrophages, displayed non-cell autonomous mechanisms in ET formation. Specifically, neurons and astroglia were indispensable for microglial ET. Macrophage colony-stimulating factor (M-CSF) secreted from these non-immune cells was essential for governing microglial ET. Neutralization of M-CSF deprived the neuron-glia conditioned medium of its ability to enable microglia to form ET when microglia encountered two lipopolysaccharide (LPS) treatments. Recombinant M-CSF protein rendered enriched microglia refractory to the second LPS challenge leading to microglial ET. Activation of microglial M-CSF receptor (M-CSFR; also known as CSF1R) and the downstream ERK1/2 signals was responsible for M-CSF-mediated microglial ET. Endotoxin-tolerant microglia in neuron-glia cultures displayed M2-like polarized phenotypes, as shown by upregulation of M2 marker Arg-1, elevated production of anti-inflammatory cytokine interleukin 10, and decreased secretion of pro-inflammatory mediators (tumor necrosis factor α, nitric oxide, prostaglandin E2 and interleukin 1β). Endotoxin-tolerant microglia protected neurons against LPS-elicited inflammatory insults, as shown by reduced neuronal damages in LPS pre-treatment group compared with the group without LPS pre-treatment. Moreover, while neurons and astroglia became injured during chronic neuroinflammation, microglia failed to form ET. Thus, this study identified a distinct non-cell autonomous mechanism of microglial ET. Interactions of M-CSF secreted by neurons and astroglia with microglial M-CSFR programed microglial ET. Loss of microglial ET could be an important pathogenetic mechanism of inflammation-associated neuronal damages.

Lymphatic Endothelial Cells Produce M-CSF, Causing Massive Bone Loss in Mice.

Gorham-Stout disease (GSD) is a rare bone disorder characterized by aggressive osteolysis associated with lymphatic vessel invasion within bone marrow cavities. The etiology of GSD is not known, and there is no effective therapy or animal model for the disease. Here, we investigated if lymphatic endothelial cells (LECs) affect osteoclasts (OCs) to cause a GSD osteolytic phenotype in mice. We examined the effect of a mouse LEC line on osteoclastogenesis in co-cultures. LECs significantly increased receptor activator of NF-κB ligand (RANKL)-mediated OC formation and bone resorption. LECs expressed high levels of macrophage colony-stimulating factor (M-CSF), but not RANKL, interleukin-6 (IL-6), and tumor necrosis factor (TNF). LEC-mediated OC formation and bone resorption were blocked by an M-CSF neutralizing antibody or Ki20227, an inhibitor of the M-CSF receptor, c-Fms. We injected LECs into the tibias of wild-type (WT) mice and observed massive osteolysis on X-ray and micro-CT scans. Histology showed that LEC-injected tibias had significant trabecular and cortical bone loss and increased OC numbers. M-CSF protein levels were significantly higher in serum and bone marrow plasma of mice given intra-tibial LEC injections. Immunofluorescence staining showed extensive replacement of bone and marrow by podoplanin+ LECs. Treatment of LEC-injected mice with Ki20227 significantly decreased tibial bone destruction. In addition, lymphatic vessels in a GSD bone sample were stained positively for M-CSF. Thus, LECs cause bone destruction in vivo in mice by secreting M-CSF, which promotes OC formation and activation. Blocking M-CSF signaling may represent a new therapeutic approach for treatment of patients with GSD. Furthermore, tibial injection of LECs is a useful mouse model to study GSD. © 2017 American Society for Bone and Mineral Research.

Inhibitory effect of neutralization of macrophage colony-stimulating factor in mechanical loading induced osteoclastogenesis and odontoclastogenesis.

Inhibitory effect of neutralization of macrophage colony-stimulating factor in mechanical loading induced osteoclastogenesis and odontoclastogenesis.

Epidermal α6β4 integrin stimulates the influx of immunosuppressive cells during skin tumor promotion

Induction of α6β4 integrin in the differentiated epidermal cell layers in skin is a hallmark of human cutaneous squamous cell carcinoma (SCC) pathogenesis and stimulates chemically induced SCC formation in Invα6β4 transgenic mice, which exhibit persistent expression of α6β4 in the suprabasal epidermal layers. However, the molecular basis for the support of SCC development by suprabasal α6β4 is not fully understood. We examined the relevance for suprabasal α6β4 expression in the epidermis for the recruitment of immunosuppressive leukocytes during the early stages of tumor promotion. In this study, we made use of the Invα6β4 transgenic mouse model, which exhibits expression of α6β4 integrin in the suprabasal layers of the epidermis driven by the involucrin promoter. First, we examined protein lysates from Invα6β4 transgenic skin using a pro-inflammatory cytokine array panel. Next, we immunofluorescence labeling of murine skin sections was employed to immunophenotype tumor promoter-treated Invα6β4 transgenic skin. Finally, a macrophage colony stimulating factor (M-CSF) neutralizing antibody strategy was administered to resolve Invα6β4 transgenic skin inflammation. Employing the Invα6β4 transgenic mouse model, we show that suprabasal α6β4 integrin expression selectively alters the profile of secreted pro-inflammatory molecules by epidermal cells, in particular CXCL5 and M-CSF, in response to acute tumor promoter treatment. The induction of CXCL5 and M-CSF in Invα6β4 transgenic epidermis was shortly followed by an exacerbated influx of CD200R(+) myeloid-derived suppressor cells (MDSCs), which co-expressed the M-CSF receptor, and FoxP3(+) Treg cells compared to wild-type mice. As a result, the levels of activated CD4(+) T lymphocytes were dramatically diminished in Invα6β4 transgenic compared to wild-type skin, whereas similar levels of lymphocyte activation were observed in the peripheral blood. Finally, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CD200R(+) infiltrative cells and epidermal proliferation were suppressed in Invα6β4 mice treated with M-CSF neutralizing antibodies. We conclude that aberrant expression of α6β4 integrin in post-mitotic epidermal keratinocytes stimulates a pro-tumorigenic skin microenvironment by augmenting the influx of immunosuppressive granular cells during tumor promotion.

Macrophage colony-stimulating factor and its receptor signaling augment glycated albumin-induced retinal microglial inflammation in vitro

BackgroundMicroglial activation and the proinflammatory response are controlled by a complex regulatory network. Among the various candidates, macrophage colony-stimulating factor (M-CSF) is considered an important cytokine. The up-regulation of M-CSF and its receptor CSF-1R has been reported in brain disease, as well as in diabetic complications; however, the mechanism is unclear. An elevated level of glycated albumin (GA) is a characteristic of diabetes; thus, it may be involved in monocyte/macrophage-associated diabetic complications.ResultsThe basal level of expression of M-CSF/CSF-1R was examined in retinal microglial cells in vitro. Immunofluorescence, real-time PCR, immunoprecipitation, and Western blot analyses revealed the up-regulation of CSF-1R in GA-treated microglial cells. We also detected increased expression and release of M-CSF, suggesting that the cytokine is produced by activated microglia via autocrine signaling. Using an enzyme-linked immunosorbent assay, we found that GA affects microglial activation by stimulating the release of tumor necrosis factor-α and interleukin-1β. Furthermore, the neutralization of M-CSF or CSF-1R with antibodies suppressed the proinflammatory response. Conversely, this proinflammatory response was augmented by the administration of M-CSF.ConclusionsWe conclude that GA induces microglial activation via the release of proinflammatory cytokines, which may contribute to the inflammatory pathogenesis of diabetic retinopathy. The increased microglial expression of M-CSF/CSF-1R not only is a response to microglial activation in diabetic retinopathy but also augments the microglial inflammation responsible for the diabetic microenvironment.

Macrophage‐colony stimulating factor as an inducer of microglial proliferation in axotomized rat facial nucleus

We analyzed the mechanism of microglial proliferation in rat axotomized facial nucleus (axotFN). In immunoblotting analysis for possible mitogens, we noticed that the amounts of macrophage-colony stimulating factor (M-CSF) increased in the axotFN for 3-7 days after transection. In contrast, the amounts of granulocyte macrophage-CSF and interleukin-3 did not significantly increase. A potential source for M-CSF was immunohistochemically verified to be microglia. Immunoblotting showed that the amounts of receptor for M-CSF (cFms) increased in the axotFN for 3-14 days after injury, and immunohistochemical staining showed that cFms is expressed in microglia. Proliferating cell nuclear antigen as a marker of proliferation was immunohistochemically identified in microglia in axotFN, and the level was found to peak 3 days after transection in immunoblotting. Hypothesizing that up-regulated M-CSF triggers the above phenomena, we investigated the effects of M-CSF on cFms and proliferating cell nuclear antigen levels in primary microglia. The biochemical experiments revealed that M-CSF induces cFms and drives the cell cycle in microglia. The neutralization of M-CSF in microglia derived from axotFN significantly reduced the proliferation. These results demonstrate that up-regulated M-CSF triggers the induction of cFms in microglia and causes the microglia to proliferate in the axotFN.

Rheumatoid synovial endothelial cells produce macrophage colony-stimulating factor leading to osteoclastogenesis in rheumatoid arthritis

Periarticular osteoporosis and joint destruction are major complications in rheumatoid arthritis (RA), caused by osteoclast-mediated bone resorption. However, the mechanisms of monocyte/osteoclast maturation and role of RA endothelial cells (RAECs) in the control of osteoclastogenesis remain unclear. The present study was designed to determine the most important factors that influence monocyte accumulation and osteoclast formation among the many factors produced by RAEC. We analysed the expression profiles of various genes in human endothelial cells from various organs (RA synovium, umbilical vein, skin, liver sinusoid, renal glomerulus and brain) using oligonucleotide microarrays. Specifically, up-regulated gene in RAECs was assessed by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and immunostaining of RA synovia. Migration of monocytes was assessed by the chemotactic chamber EZ-TAXIScan. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation was observed by microscopy. Among many epithelial-expressed factors, macrophage colony-stimulating factor (M-CSF) gene was abundantly expressed specifically in RAECs. Genes of fibroblast growth factor-2, interleukin-6 and osteoprotegerin were also overexpressed in RAECs. Migration of monocytes and osteoclast formation in co-cultures promoted by culture supernatants of RAECs were inhibited by M-CSF neutralizing antibody. M-CSF produced by RAECs is involved in osteoclastogenesis from monocytes, migration and TRAP-positive MNC formation.

Tumor Necrosis Factor-α Increases Circulating Osteoclast Precursor Numbers by Promoting Their Proliferation and Differentiation in the Bone Marrow through Up-regulation of c-Fms Expression

Osteoclasts are essential cells for bone erosion in inflammatory arthritis and are derived from cells in the myeloid lineage. Recently, we reported that tumor necrosis factor-alpha (TNFalpha) increases the blood osteoclast precursor (OCP) numbers in arthritic patients and animals, which are reduced by anti-TNF therapy, implying that circulating OCPs may have an important role in the pathogenesis of erosive arthritis. The aim of this study is to investigate the mechanism by which TNFalpha induces this increase in OCP frequency. We found that TNFalpha stimulated cell division and conversion of CD11b+/Gr-1-/lo/c-Fms- to CD11b+/Gr-1-/lo/c-Fms+ cells, which was not blocked by neutralizing macrophage colony-stimulating factor (M-CSF) antibody. Ex vivo analysis of monocytes demonstrated the following: (i) blood CD11b+/Gr-1-/lo but not CD11b-/Gr-1- cells give rise to osteoclasts when they were cultured with receptor activator NF-kappaB ligand and M-CSF; and (ii) TNF-transgenic mice have a significant increase in blood CD11b+/Gr-1-/lo cells and bone marrow proliferating CD11b+/Gr-1-/lo cells. Administration of TNFalpha to wild type mice induced bone marrow CD11b+/Gr-1-/lo cell proliferation, which was associated with an increase in CD11b+/Gr-1-/lo OCPs in the circulation. Thus, TNFalpha directly stimulates bone marrow OCP genesis by enhancing c-Fms expression. This results in progenitor cell proliferation and differentiation in response to M-CSF, leading to an enlargement of the marrow OCP pool. Increased marrow OCPs subsequently egress to the circulation, forming a basis for elevated OCP frequency. Therefore, the first step of TNF-induced osteoclastogenesis is at the level of OCP genesis in the bone marrow, which represents another layer of regulation to control erosive disease.

Macrophage colony stimulating factor promotes phagocytosis by murine microglia

Macrophage colony stimulating factor (M-CSF) and its receptor are upregulated in the brain in Alzheimer's disease. M-CSF induces activation and proliferation of microglial cells and expression of proinflammatory cytokines. Amyloid beta (Aβ) immunization experiments suggest that microglia have the capacity to aggressively clear Aβ from the brain under certain circumstances. We examined the role of M-CSF in phagocytosis of fluorescent microspheres and Aβ by cultured microglia. M-CSF treatment increased microglial cell phagocytosis of both microspheres and of Aβ. Antibody neutralization of M-CSF inhibited Aβ uptake induced by overexpression of the M-CSF receptor on microglia. These results suggest that M-CSF could be important in promoting microglial clearance of abnormal protein aggregates such as Aβ.

Overexpression of Macrophage Colony-stimulating Factor Receptor on Microglial Cells Induces an Inflammatory Response

Microglia are important in the inflammatory response in Alzheimer's disease (AD). We showed previously that macrophage colony-stimulating factor receptor (M-CSFR), encoded by the c-fms protooncogene, is overexpressed on microglia surrounding amyloid beta (Abeta) deposits in the APP(V717F) mouse model for AD. The M-CSFR is also increased on microglia after experimental brain injury and in AD. To determine the relevance of these findings, we transiently expressed M-CSFR on murine BV-2 and human SV-A3 microglial cell lines using an SV40-promoted c-fms construct. M-CSFR overexpression resulted in microglial proliferation and increased expression of inducible nitric-oxide synthase, the proinflammatory cytokines interleukin-1alpha, macrophage inflammatory protein 1-alpha, and interleukin-6 and of macrophage colony-stimulating factor (M-CSF) itself. Antibody neutralization of M-CSF showed that the M-CSFR-induced proinflammatory response was dependent on M-CSF in the culture media. By using a co-culture of c-fms-transfected murine microglia and rat organotypic hippocampal slices and a species-specific real time reverse transcriptase-polymerase chain reaction assay and enzyme-linked immunosorbent assay, we showed that M-CSFR overexpression on exogenous microglia induced expression of interleukin-1alpha by the organotypic culture. These results show that increased M-CSFR expression induces microglial proliferation, cytokine expression, and a paracrine inflammatory response, suggesting that in APP(V717F) mice increased M-CSFR on microglia could be an important factor in Abeta-induced inflammatory response.

Cytokine-regulated expression of activated leukocyte cell adhesion molecule (CD166) on monocyte-lineage cells and in rheumatoid arthritis synovium.

To determine whether monocyte/macrophage expression of the CD6 ligand, activated leukocyte cell adhesion molecule (ALCAM) (CD166), is regulated by cytokines during inflammation in rheumatoid arthritis (RA). We used flow cytometry to test whether cytokines present in rheumatoid synovium could regulate ALCAM cell surface expression on peripheral blood (PB) monocytes and RA synovial fluid (SF) macrophages, and we examined ALCAM expression in situ in RA synovium by immunofluorescence. The monocyte differentiation factors interleukin-3, macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor augmented ALCAM expression on PB monocytes. ALCAM was expressed on monocyte-lineage cells in situ in inflamed synovium from patients with RA (9 of 9), but not in uninflamed synovium from patients with joint trauma (0 of 3). Furthermore, in vitro culture-induced ALCAM expression on PB monocytes and CD14+ RA SF cells was inhibited by an M-CSF neutralizing antibody. ALCAM expression on PB and SF monocytes/macrophages is enhanced by M-CSF.