Nanotopographical Coatings Induce an Early Phenotype-Specific Response of Primary Material-Resident M1 and M2 Macrophages.
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ABSTRACT:
SUBMITTER: Schmitz T
PROVIDER: S-EPMC7084960 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Older adults suffer from weakened and delayed bone healing due to age-related alterations in bone cells and in the immune system. Given the interaction between the immune system and skeletal cells, therapies that address deficiencies in both the skeletal and the immune system are required to effectively treat bone injuries of older patients. The sequence of macrophage activation observed in healthy tissue repair involves a transition from a pro-inflammatory state followed by a pro-reparative state. In older patients, inflammation is slower to resolve and impedes healing. The goal of this study was to design a novel drug delivery system for temporal guidance of the polarization of macrophages using bone grafting materials. A biomimetic calcium phosphate coating (bCaP) physically and temporally separated the pro-inflammatory stimulus interferon-gamma (IFN?) from the pro-reparative stimulus simvastatin (SIMV). Effective doses were identified using a human monocyte line (THP-1) and testing culminated with bone marrow macrophages obtained from old mice. Sequential M1-to-M2 activation was achieved with both cell types. These results suggest that this novel immunomodulatory drug delivery system holds potential for controlling macrophage activation in bones of older patients.
| S-EPMC6336526 | biostudies-literature
Project description:BackgroundToll-like receptors (TLRs) and macrophages play an important role in rheumatoid arthritis (RA). Currently, it is not clear whether inflammatory M1 or anti-inflammatory M2 predominate among the resident macrophages in the synovium. In the present study, we set out to investigate the impact of TLR stimulation on monocyte-derived M1 and M2 macrophage function and phenotype by mimicking the exposure to abundant TLR agonists as occurs in the context of RA. The response of macrophage subsets to TLR2 and TLR4 activation was evaluated on cluster of differentiation (CD) marker profile; cytokine secretion; gene expression; and NF-?B, interferon regulatory factors 3 and 7 (IRF3/7), and mitogen-activated protein kinase (MAPK) activation.MethodsHuman monocytes were isolated from peripheral blood of healthy individuals and patients with RA and differentiated into M1-like and M2-like macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Cells were either (1) stimulated with TLR ligands Pam3 or lipopolysaccharide (LPS) or (2) classically activated via interferon (IFN)-?/LPS. Cytokine production was measured by enzyme-linked immunosorbent assay, and gene expression was measured by qPCR. Cells were stained for CD markers and analyzed by fluorescence-activated cell sorting. NF-?B, IRF3/7, and MAPKs were detected by Western blotting.ResultsMonocyte-derived macrophages of healthy donors (HD) or patients with RA displayed comparable subset-specific phenotypes upon exposure to TLR agonists. CD14 and CD163 marker expression on M2 macrophages did not change upon TLR2 and TLR4 engagement. By contrast, M2 gene markers HMOX1, FOLR2, and SLC40A1 were decreased. Importantly, M2 macrophages derived from HD or patients with RA showed both a decreased ratio of interleukin (IL)-10/IL-6 and IL-10/IL-8 upon stimulation with TLR2 ligand Pam3 compared with TLR4 ligand LPS. Gene expression of TLR2 was increased, whereas TLR4 expression was decreased, by TLR ligand stimulation. MAPKs p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase were activated more strongly in M2 than in M1 macrophages by Pam3 or LPS.ConclusionsWe show that the anti-inflammatory activity of M2 macrophages is reduced in the presence of abundant TLR2 ligands without significant changes in cell surface markers. Thus, the classical M1/M2 paradigm based on cellular markers does not apply to macrophage functions in inflammatory conditions such as RA.
| S-EPMC5667453 | biostudies-literature
Project description:Inflammation is pivotal in atherosclerosis. Monocyte-macrophages are crucial in atherosclerosis. Monocytes can develop into subsets: classically (M1) or alternatively (M2) activated cells. Several studies point to a proinflammatory role for C-reactive protein (CRP). Because there is a paucity of data on the effects of CRP on macrophage phenotype, we tested effects of CRP on macrophage polarization.Monocytes were incubated with CRP (0 to 50 ?g/mL) and differentiated into macrophages for 7 days. Phenotypic characterization of M1 and M2 macrophages was performed using flow cytometry. CRP treatment resulted in increased population of M1 macrophages (tumor necrosis factor [TNF]/interleukin [IL]-12/C-C chemokine receptor 2, TNF/IL-12/monocyte chemotactic protein-1, or TNF/IL-1/IL-12). These effects were not abrogated by polymixin B or small interfering RNA to Toll-like receptor-4, but they were abrogated by boiled CRP. Administration of human CRP to rats in vivo increased polarization of macrophages to M1 phenotype compared with human serum albumin. When macrophages were primed to the M2 phenotype with IL-4, addition of CRP resulted in significantly increased secretion of TNF-?, MCP-1, and IL-1 and conversion of macrophages from the M2 to the M1 phenotype. CRP failed to prime macrophages to the M1 phenotype in presence of CD32/CD64 small interfering RNA or dominant-negative nuclear factor kappa B.Collectively, these results further support a role for CRP in promoting differentiation of human monocytes toward a proinflammatory M1 phenotype.
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Project description:Patients with chronic anterior uveitis are at particularly high risk of developing secondary glaucoma when corticosteroids [e.g., dexamethasone (Dex)] are used or when inflammatory activity has regressed. Macrophage migration into the eye increases when secondary glaucoma develops and may play an important role in the development of secondary glaucoma. Our aim was to evaluate in vitro if increased hydrostatic pressure and corticosteroids could induce changes in macrophages phenotype. By using a pressure chamber cell culture system, we assessed the effect of increased hydrostatic pressure (HP), inflammation, and immunosuppression (Dex) on the M1/M2 phenotype of macrophages. Bone marrow-derived macrophages (BMDMs) were stimulated with medium, lipopolysaccharide (LPS, 100 ng/ml), Dex (200 ng/ml), or LPS + Dex and incubated with different HP (0, 20, or 60 mmHg) for 2 or 7 days. The numbers of CD86+/CD206- (M1 phenotype), CD86-/CD206+ (M2 phenotype), CD86+/CD206+ (intermediate phenotype), F4/80+/TNF-?+, and F4/80+/IL-10+ macrophages were determined by flow cytometry. TNF-? and IL-10 levels in cell culture supernatants were quantified by ELISA. TNF-?, IL-10, fibronectin, and collagen IV expression in BMDMs were detected by immunofluorescence microscopy. Higher HP polarizes macrophages primarily to an M1 phenotype (LPS, 60 vs. 0 mmHg, d2: p = 0.0034) with less extra cellular matrix (ECM) production and secondary to an M2 phenotype (medium, 60 vs. 0 mmHg, d7: p = 0.0089) (medium, 60 vs. 20 mmHg, d7: p = 0.0433) with enhanced ECM production. Dex induces an M2 phenotype (Dex, medium vs. Dex, d2: p < 0.0001; d7: p < 0.0001) with more ECM production. Higher HP further increased M2 polarization of Dex-treated macrophages (Dex, 60 vs. 0 mmHg, d2: p = 0.0417; d7: p = 0.0454). These changes in the M1/M2 phenotype by high HP or Dex treatment may play a role in the pathogenesis of secondary uveitic glaucoma- or glucocorticoid (GC)-induced glaucoma.
| S-EPMC7591771 | biostudies-literature
Project description:Macrophages are multifunctional cells that perform diverse roles in health and disease and considered the main source of inflammatory cytokines in affected joints of patients with rheumatoid arthritis (RA). M2 macrophages are well known as anti-inflammation and wound-healing cells; however, recent evidence suggests that they can also promote inflammation in RA, although the underlying mechanism remains to be clarified. Based upon our recent finding that lactoferrin (LTF)-containing IgG immunocomplex (LTF-IC), found elevated in RA sera, potent activators of human monocytes/macrophages, we herein demonstrate that LTF-IC was able to elicit immediate proinflammatory cytokine production by M2-polarized human macrophages through coligation with CD14/toll-like receptor (TLR) 4 and FcγRIIa (CD32a). The LTF-IC-treated M2 cells adopted surface maker expression profile similar to that of M1 phenotype and became functionally hyperactive to subsequent stimuli such as lipopolysaccharide, zymosan and IL-1β, which could provide a positive feedback signal to promote excessive inflammation in RA. They also acquired the ability to facilitate activation of Th17 cells that are known to play critical roles in RA pathology. We propose that IgG ICs containing TLR agonizing autoantigens are able to directly switch human macrophages from M2 into M1-like phenotype, thereby promoting excessive inflammation in autoimmune diseases such as RA.
| S-EPMC5787126 | biostudies-literature
Project description:Macrophages become polarized by cues in their environment and this polarization causes a functional change in their behavior. Two main subsets of polarized macrophages have been described. M1, or "classically activated" macrophages, are pro-inflammatory and M2, or "alternatively activated" macrophages, are anti-inflammatory. In this study, we investigated the motility and force generation of primary human macrophages polarized down the M1 and M2 pathways using chemokinesis assays and traction force microscopy on polyacrylamide gels. We found that M1 macrophages are significantly less motile and M2 macrophages are significantly more motile than unactivated M0 macrophages. We also showed that M1 macrophages generate significantly less force than M0 or M2 macrophages. We further found that M0 and M2, but not M1, macrophage force generation is dependent on ROCK signaling, as identified using the chemical inhibitor Y27632. Finally, using the chemical inhibitor blebbistatin, we found that myosin contraction is required for force generation by M0, M1, and M2 macrophages. This study represents the first investigation of the changes in the mechanical motility mechanisms used by macrophages after polarization.
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Project description:Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages.
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Project description:Macrophages are critical mediators of the innate immune response against foreign pathogens, including bacteria, physical stress, and injury. Therefore, these cells play a key role in the "inflammatory pathway" which in turn can lead to an array of diseases and disorders such as autoimmune neuropathies and myocarditis, inflammatory bowel disease, atherosclerosis, sepsis, arthritis, diabetes, and angiogenesis. Recently, more studies have focused on the macrophages inflammatory diseases since the discovery of the two subtypes of macrophages, which are differentiated on the basis of their phenotype and distinct gene expression pattern. Of these, M1 macrophages are pro-inflammatory and responsible for inflammatory signaling, while M2 are anti-inflammatory macrophages that participate in the resolution of the inflammatory process, M2 macrophages produce anti-inflammatory cytokines, thereby contributing to tissue healing. Many studies have shown the role of these two subtypes in the inflammatory pathway, and their emergence appears to decide the fate of inflammatory signaling and disease progression. As a next step in directing the pro-inflammatory response toward the anti-inflammatory type after an insult by a foreign pathogen (e. g., bacterial lipopolysaccharide), investigators have identified many natural compounds that have the potential to modulate M1 to M2 macrophages. In this review, we provide a focused discussion of advances in the identification of natural therapeutic molecules with anti-inflammatory properties that modulate the phenotype of macrophages from M1 to M2.
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Project description:Macrophages, differentiation from monocytes infiltrated in the wound, have been suggested to be involved and to play an important role in the pathogenesis of wound healing. Nevertheless, no evidence has been established regarding M1 and M2 type macrophages in Keloid. To understand the status of M1 and M2 type macrophages in keloid, immunohistochemistry was performed on 30 cases of Keloid tissues and normal controls, with CD68, typical surface marker for M1 and CD163, well-accepted marker for M2 being immunostained. Meanwhile, the glucocorticoid receptor NR3C1 was also detected. As further confirmation, quantitative real-time PCR was utilized to verify the expression of CD68, CD163 and NR3C1 on mRNA level. It was consistently shown that infiltrated M2 macrophages pronouncedly outnumbered M1 macrophages in the dermis of keloids; and that NR3C1 expression was significantly up-regulated in keloids than that in normal controls. In addition, there was a marked correlation between CD163 and NR3C1 expression. Our results suggest that the number of infiltrated M2 macrophages in the dermis of keloids may be linked to the responsiveness to glucocorticoids in the pathogenesis of keloid.
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Project description:Pleural macrophages play critical roles in pathogenesis of tuberculous pleuritis, but very little is known about their response to anti-tuberculosis antibiotics treatment. Here, we examined whether and how pleural macrophages change in phenotype, transcription and function following antibiotics treatment in patients with tuberculous pleuritis. Results show pro-inflammatory cytokines were down-regulated significantly post antibiotic treatment in the pleural effusions and pleural macrophages up-regulated markers characteristic of M2 macrophages such as CD163 and CD206. Differential expression analysis of transcriptomes from four paired samples before and after treatment identified 230 treatment-specific responsive genes in pleural macrophages. Functional analysis identified interferon-related pathway to be the most responsive genes and further confirmed macrophage polarization to M2-like phenotype. We further demonstrate that expression of a significant fraction of responsive genes was modulated directly by antibiotics in pleural macrophages in vitro. Our results conclude that pleural macrophages polarize from M1-like to M2-like phenotype within a mean of 3.5 days post antibiotics treatment, which is dependent on both pleural cytokine environment and direct modulatory effects of antibiotics. The treatment-specific genes could be used to study the roles of pleural macrophages in the pathogenesis of tuberculous pleuritis and to monitor the response to antibiotics treatment.
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