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CCL2, CCL5, and IGF-1 participate in the immunomodulation of osteogenesis during M1/M2 transition in vitro.

ABSTRACT: The modulation of macrophage phenotype from pro-inflammatory (M1) to tissue healing (M2) via exogenous addition of interleukin-4 (IL-4) facilitates osteogenesis; however, the molecular mediators underlying this phenomenon remain unknown. This study characterizes the IL-4-dependent paracrine crosstalk between macrophages and osteoprogenitors and its effect on osteogenesis in vitro. Primary murine M1 were co-cultured with MC3T3 cells (M1-MC3T3) in both transwell plates and direct co-cultures. To modulate M1 to M2, M1-MC3T3 were treated with IL-4 (20 ng/mL) at day 3 after seeding (M1?+?IL-4-MC3T3). Selected molecular targets were assessed at days 3 and 6 after seeding at protein and mRNA levels. Mineralization was assessed at day 21. Transwell M1?+?IL-4-MC3T3 significantly enhanced the secretion of CCL2/MCP-1, IGF-1 and to a lesser degree, CCL5/RANTES at day 6. At day 3, alkaline phosphatase (Alpl) was upregulated in direct M1-MC3T3. At day 6, Smurf2 and Insulin growth factor-1 (IGF-1) were downregulated and upregulated, respectively, in direct M1?+?IL-4-MC3T3. Finally, M1?+?IL-4-MC3T3 increased bone matrix mineralization compared with MC3T3 cells in transwell, but this was significantly less than M1-MC3T3. Taken together, macrophage subtypes enhanced the osteogenesis in transwell setting and the transition from M1 to M2 was associated with an increase in bone anabolic factors CCL2/MCP-1, CCL5/RANTES and IGF-1 in vitro. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3069-3076, 2017.

SUBMITTER: Cordova LA

PROVIDER: S-EPMC5748268 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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CCL2, CCL5, and IGF-1 participate in the immunomodulation of osteogenesis during M1/M2 transition in vitro.

Córdova Luis A LA Loi Florence F Lin Tzu-Hua TH Gibon Emmanuel E Pajarinen Jukka J Nabeshima Akira A Lu Laura L Yao Zhenyu Z Goodman Stuart B SB

Journal of biomedical materials research. Part A 20170821 11

The modulation of macrophage phenotype from pro-inflammatory (M1) to tissue healing (M2) via exogenous addition of interleukin-4 (IL-4) facilitates osteogenesis; however, the molecular mediators underlying this phenomenon remain unknown. This study characterizes the IL-4-dependent paracrine crosstalk between macrophages and osteoprogenitors and its effect on osteogenesis in vitro. Primary murine M1 were co-cultured with MC3T3 cells (M1-MC3T3) in both transwell plates and direct co-cultures. To m ...[more]

PMID: 28782174

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Project description:BackgroundBone formation and remodeling are influenced by the inflammatory state of the local microenvironment. In this regard, macrophages are postulated to play a crucial role in modulating osteogenesis. However, the differential effects of macrophage subsets and their plasticity on bone formation are currently unknown.MethodsPolarized primary murine macrophages and preosteoblastic MC3T3 cells were co-cultured to investigate the effect of non-activated M0, pro-inflammatory M1, and tissue-regenerative M2 macrophages on the osteogenic ability of MC3T3-E1 cells in vitro. Furthermore, to model the physiological transition from inflammation to tissue regeneration, M1-MC3T3 co-cultures were treated with interleukin-4 (IL-4) at different time points to modulate the M1 phenotype towards M2. Macrophage phenotypic markers were assessed by flow cytometry and enzyme-linked immunosorbent assay. A time course study of osteogenic markers at different time points was conducted: alkaline phosphatase (ALP) mRNA levels were evaluated at week 1, ALP activity and osteocalcin and osteopontin mRNA levels at week 2, and matrix mineralization and osteocalcin and osteopontin protein concentrations at week 3. Supernatant collected 72 hours after seeding or IL-4 treatment, whichever was later, was analyzed for oncostatin M, a cytokine released by macrophages that has been recognized to enhance osteogenesis. Unpaired t test or one-way ANOVA with Tukey's or Dunnett's post hoc tests were used for statistical comparison of the groups.ResultsCo-culture with any of the macrophage subtypes increased the osteogenic ability of MC3T3 cells as indicated by increases in ALP activity and matrix mineralization. Increased ALP activity, osteocalcin concentration, and matrix mineralization demonstrated that osteogenesis by M1-MC3T3 co-cultures was further enhanced by macrophage phenotype modulation to M2 via IL-4 treatment 72 hours after seeding. Increased oncostatin M protein concentration in untreated M1-MC3T3 co-cultures and M1-MC3T3 co-cultures treated with IL-4 at 72 hours correlated with greater ALP activity and matrix mineralization.ConclusionsThese results suggest that a transient inflammatory phase is crucial for enhanced bone formation. Macrophage plasticity may offer new strategies for modulating the local inflammatory microenvironment with the aim of potentially enhancing bone repair.

Project description:BackgroundThe inflammatory chemokines CCL2 (MCP-1) & CCL5 (RANTES) and the inflammatory cytokines TNF? & IL-1? were shown to contribute to breast cancer development and metastasis. In this study, we wished to determine whether there are associations between these factors along stages of breast cancer progression, and to identify the possible implications of these factors to disease course.MethodsThe expression of CCL2, CCL5, TNF? and IL-1? was determined by immunohistochemistry in patients diagnosed with: (1) Benign breast disorders (=healthy individuals); (2) Ductal Carcinoma In Situ (DCIS); (3) Invasive Ducal Carcinoma without relapse (IDC-no-relapse); (4) IDC-with-relapse. Based on the results obtained, breast tumor cells were stimulated by the inflammatory cytokines, and epithelial-to-mesenchymal transition (EMT) was determined by flow cytometry, confocal analyses and adhesion, migration and invasion experiments.ResultsCCL2, CCL5, TNF? and IL-1? were expressed at very low incidence in normal breast epithelial cells, but their incidence was significantly elevated in tumor cells of the three groups of cancer patients. Significant associations were found between CCL2 & CCL5 and TNF? & IL-1? in the tumor cells in DCIS and IDC-no-relapse patients. In the IDC-with-relapse group, the expression of CCL2 & CCL5 was accompanied by further elevated incidence of TNF? & IL-1? expression. These results suggest progression-related roles for TNF? and IL-1? in breast cancer, as indeed indicated by the following: (1) Tumors of the IDC-with-relapse group had significantly higher persistence of TNF? and IL-1? compared to tumors of DCIS or IDC-no-relapse; (2) Continuous stimulation of the tumor cells by TNF? (and to some extent IL-1?) has led to EMT in the tumor cells; (3) Combined analyses with relevant clinical parameters suggested that IL-1? acts jointly with other pro-malignancy factors to promote disease relapse.ConclusionsOur findings suggest that the coordinated expression of CCL2 & CCL5 and TNF? & IL-1? may be important for disease course, and that TNF? & IL-1? may promote disease relapse. Further in vitro and in vivo studies are needed for determination of the joint powers of the four factors in breast cancer, as well as analyses of their combined targeting in breast cancer.

Project description:Novel minimally invasive strategies are needed to obtain robust bone healing in complex fractures and bone defects in the elderly population. Local cell therapy is one potential option for future treatment. Mesenchymal stromal cells (MSCs) are not only involved in osteogenesis but also help direct the recruitment of macrophages during bone regeneration via MSC-macrophage crosstalk. The C-C motif chemokine ligand 2 (CCL2) is an inflammatory chemokine that is associated with the migration of macrophages and MSCs during inflammation. This study investigated the use of CCL2 as a therapeutic target for local cell therapy. MSCs and macrophages were isolated from 10 to 12 week-old BALB/c male mice. Genetically modified CCL2 over-expressing MSCs were produced using murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector. Osteogenic differentiation assays were performed using MSCs with or without macrophages in co-culture. Cell migration assays were also performed. MSCs transfected with murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector showed higher levels of CCL2 secretion compared to unaltered MSCs (p < 0.05). Genetic manipulation did not affect cell proliferation. CCL2 did not affect the osteogenic ability of MSCs alone. However, acute (1 day) but not sustained (7 days) stimulation with CCL2 increased the alizarin red-positive area when MSCs were co-cultured with macrophages (p < 0.001). Both recombinant CCL2 (p < 0.05) and CCL2 released from MSCs (p < 0.05) facilitated macrophage migration. We demonstrated that acute CCL2 stimulation promoted subsequent osteogenesis in co-culture of MSCs and macrophages. Acute CCL2 stimulation potentially facilitates osteogenesis during the acute inflammatory phase of bone healing by directing local macrophage migration, fostering macrophage-MSC crosstalk, and subsequently, by activating or licensing of MSCs by macrophage pro-inflammatory cytokines. The combination of CCL2, MSCs, and macrophages could be a potential strategy for local cell therapy in compromised bone healing.

Project description:BackgroundTreatment for neuropathic pain as a refractory disease remains unsatisfactory and represents a significant clinical challenge. A highly effective drug is thus urgently needed for neuropathic pain treatment. Dihydromyricetin (DMY) is a flavonoid with a wide range of biological activities. The purpose of this research is to explore the effects of DMY on neuropathic pain and the underlying mechanism of its effect.MethodsThe effect of DMY was investigated in BV-2 cells and lipopolysaccharide (LPS)-induced BV-2 cells. A neuropathic pain model was established via spared nerve injury (SNI) surgery in mice, and the protein expression level was detected via Western blot assay. The percent of M1 and M2 phenotype polarization cells were detected via flow cytometry assay. Immunochemical staining assay was also performed to measure the marker levels of the M1 and M2 phenotype polarization cells and aldehyde dehydrogenase 2 (ALDH2) level, and mechanical pain sensitivity was evaluated via measurement of the mechanical withdrawal threshold.ResultsWe found that DMY promoted the transition from M1 to M2 polarization and upregulated the ALDH2 level in vitro and vitro. ALDA-1, an ALDH2 agonist, promoted the switching from M1 to M2 polarization in vivo and vitro. DMY alleviated pain hypersensitivity induced by SNI via enhancing M2 phenotype polarization by elevating ALDH2 activity in mice. After DMY- or ALDA-1-microglia were injected into SNI-induced pain hypersensitive mice, the mechanical withdrawal threshold was increased significantly when compared with the SNI group.ConclusionsOur data demonstrated that DMY alleviated neuropathic pain via enhancing the polarization transition from the M1 to M2 phenotype by potentially elevating ALDH2 activity in vitro and vivo. DMY- or ALDA-1-microglia may have alleviative effects on neuropathic pain. The findings herein provide a promising avenue for neuropathic pain treatment, suggesting a new target, ALDH2, in the treatment of neuropathic pain.

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CCL2, CCL5, and IGF-1 participate in the immunomodulation of osteogenesis during M1/M2 transition in vitro.

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CCL2, CCL5, and IGF-1 participate in the immunomodulation of osteogenesis during M1/M2 transition in vitro.

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