Macrophage differentiation and polarization on a decellularized pericardial biomaterial.
ABSTRACT: The monocyte-derived macrophage (MDM), present at biomaterial implantations, can increase, decrease or redirect the inflammatory and subsequent wound healing process associated with the presence of a biomaterial. Understanding MDM responses to biomaterials is important for improved prediction and design of biomaterials for tissue engineering. This study analyzed the direct differentiation of monocytes on intact, native collagen. Human monocytes were differentiated on decellularized bovine pericardium (DBP), polydimethylsiloxane (PDMS) or polystyrene (TCPS) for 14 d. MDMs on all surfaces released high amounts of MMP-9 compared to MMP-2 and relatively little MMP-1. MDMs differentiated on DBP released more MMP-2, but less acid phosphatase activity. MDMs on all three surfaces released low amounts of cytokines, although substrate differences were found: MDMs on DBP released higher amounts of IL-6, IL-8, and MCP-1 but lower amounts of IL-10 and IL-1ra. This research provides evidence that MDMs on decellularized matrices may not be stimulated towards an activated, inflammatory phenotype, supporting the potential of decellularized matrices for tissue engineering. This study also demonstrated that the differentiation surface affects MDM phenotype and therefore study design of macrophage interactions with biomaterials should scrutinize the specific macrophage culture method utilized and its effects on macrophage phenotype.
Project description:The prevalence of a macrophage phenotype in atherosclerotic plaque may drive its progression and/or instability. Macrophages from coronary plaques are not available, and monocyte-derived macrophages (MDMs) are usually considered as a surrogate. We compared the MDM profile obtained from coronary artery disease (CAD) patients and healthy subjects, and we evaluated the association between CAD MDM profile and in vivo coronary plaque characteristics assessed by optical coherence tomography (OCT). At morphological analysis, MDMs of CAD patients had a higher prevalence of round than spindle cells, whereas in healthy subjects the prevalence of the two morphotypes was similar. Compared to healthy subjects, MDMs of CAD patients had reduced efferocytosis, lower transglutaminase-2, CD206 and CD163 receptor levels, and higher tissue factor (TF) levels. At OCT, patients with a higher prevalence of round MDMs showed more frequently a lipid-rich plaque, a thin-cap fibroatheroma, a greater intra-plaque macrophage accumulation, and a ruptured plaque. The MDM efferocytosis correlated with minimal lumen area, and TF levels in MDMs correlated with the presence of ruptured plaque. MDMs obtained from CAD patients are characterized by a morpho-phenotypic heterogeneity with a prevalence of round cells, showing pro-inflammatory and pro-thrombotic properties. The MDM profile allows identifying CAD patients at high risk.
Project description:<h4>Background</h4>Organic dust exposure in the agricultural industry results in significant airway disease and lung function decrease. Mononuclear phagocytes are key cells that mediate the inflammatory and innate immune response after dust exposure.<h4>Objective</h4>We sought to investigate the effect of organic dust extract (ODE) from modern swine operations on monocyte-derived macrophage (MDM) phenotype and function.<h4>Methods</h4>Peripheral blood monocytes were obtained by means of elutriation methodology (>99% CD14(+)) and differentiated into macrophages in the presence of GM-CSF (1 week) with and without ODE (0.1%). At 1 week, cells were analyzed by means of flow cytometry for cell-surface marker expression (HLA-DR, CD80, CD86, Toll-like receptor 2, Toll-like receptor 4, mCD14, and CD16), phagocytosis (IgG-opsonized zymosan particles), and intracellular killing of Streptococcus pneumoniae. At 1 week, MDMs were rechallenged with high-dose ODE (1%), LPS, and peptidoglycan (PGN), and cytokine levels (TNF-alpha, IL-6, IL-10, and CXCL8/IL-8) were measured. Comparisons were made to MDMs conditioned with heat-inactivated dust, endotoxin-depleted dust, LPS, and PGN to elucidate ODE-associated factors.<h4>Results</h4>Expression of HLA-DR, CD80, and CD86; phagocytosis; and intracellular bacterial killing were significantly decreased with ODE-challenged versus control MDMs. Responses were retained after marked depletion of endotoxin. PGN, LPS, and PGN plus LPS significantly reduced MDM surface marker expression and, except for LPS alone, also reduced phagocytosis. ODE-challenged MDMs had significantly diminished cytokine responses (TNF-alpha, IL-6, and IL-10) after repeat challenge with high-dose ODE. Cross-tolerant cytokine responses were also observed.<h4>Conclusion</h4>Repetitive organic dust exposure significantly decreases markers of antigen presentation and host defense function in MDMs. Bacterial cell components appear to be driving these impaired responses.
Project description:Circulating monocytes recruited to tissues can differentiate into macrophages and adopt unique gene expression programs in response to environmental cues. We recently described the regulated expression of several microRNAs (miRNAs) in polarized human monocyte-derived macrophages (MDMs). Basal expression of these activation-associated miRNAs was low in monocytes relative to MDMs. As development occurs in the context of specific cellular environments, we hypothesized that the rate of miRNA accumulation would be modified in the presence of microbial or cellular products during monocyte-to-macrophage differentiation. Indeed, LPS treatment augmented the accumulation of miR-146a and miR-155, whereas IL-4 treatment augmented the accumulation of miR-193b and miR-222 during development. In contrast, some stimuli repressed accumulation of specific miRNAs including interferons (IFNs) (miR-27a, miR-125a-5p, and miR-222), IL-4 (miR-125a-5p), and LPS (miR-27a). RT-PCR-based expression profiling of monocytes differentiated with distinct methods showed that activation-associated miRNAs and markers of macrophage polarization were substantially altered in MDMs differentiated in the presence of non-monocytic peripheral blood mononuclear cells due in part to NF-?B and STAT1 pathway activation. Expression of several of these miRNAs was regulated at a preprocessing step because the expression of the primary miRNAs, but not Dicer, correlated with mature miRNA expression. We conclude that a set of miRNAs is regulated during MDM differentiation, and the rate is uniquely modified for each miRNA by environmental factors. The low basal expression of activation-associated miRNAs in monocytes and their dynamic rates of accumulation during MDM differentiation permit monocytes to tailor miRNA profiles in peripheral tissues during differentiation to macrophages.
Project description:Macrophages play a crucial role in the biological performance of biomaterials, as key factors in defining the optimal inflammation-healing balance towards tissue regeneration and implant integration. Here, we investigate how different surface modifications performed on poly(l-lactic acid) (PLLA) films would influence the differentiation of human monocytes into macrophages. We tested PLLA films without modification, surface-modified by plasma treatment (pPLLA) or by combining plasma treatment with different coating materials, namely poly(l-lysine) and a series of proteins from the extracellular matrix: collagen I, fibronectin, vitronectin, laminin and albumin. While all the tested films are non-cytotoxic, differences in cell adhesion and morphology are observed. Monocyte-derived macrophages (MDM) present a more rounded shape in non-modified films, while a more elongated phenotype is observed containing filopodia-like and podosome-like structures in all modified films. No major differences are found for the expression of HLA-DR+/CD80+ and CD206+/CD163+ surface markers, as well as for the ability of MDM to phagocytize. Interestingly, MDM differentiated on pPLLA present the highest expression of MMP9. Upon differentiation, MDM in all surface modified films present lower amounts of IL-6 and IL-10 compared to non-modified films. After stimulating MDM with the potent pro-inflammatory agent LPS, pPLLA and poly(l-lysine) and fibronectin-modified films reveal a significant reduction in IL-6 secretion, while the opposite effect is observed with IL-10. Of note, in comparison to non-modified films, all surface modified films induce a significant reduction of the IL-6/IL-10 ratio, a valuable prognosticator of the pro- versus anti-inflammatory balance. These findings provide important insights into MDM-biomaterial interactions, while strengthening the need for designing immune-informed biomaterials.
Project description:To study distinct aspects of human monocyte-derived macrophage (MDM) activation by human corneal tissue as a possible initial stage in human corneal allograft rejection.Human monocytes were isolated from peripheral blood mononuclear cells (PBMC) and differentiated into MDM. Human corneas with or without endothelium were fragmented using a standardized protocol. MDM were stimulated with human corneal fragments, corneal fragment supernatant, lipopolysaccharide (LPS) or interferon-gamma (IFN?), and expression profiles for 34 cytokines were determined in MDM-conditioned media using a Luminex bead-based multiplex assay. Data from clinical aqueous humour samples served for comparison and validation. To assess cell recruitment, immunogenicity of corneal endothelial cells (CEC), monocyte survival and differentiation, we applied transwell migration assays, cell viability assays and fluorescence-activated cell sorting, respectively.Corneal fragments induced MDM to release distinct cytokines into the medium. Media thus conditioned in vitro by stimulated MDM shared cytokine patterns, namely MCP-1, MIP-1? and MIP-1?, with human aqueous humor samples obtained in human corneal allograft rejection. The presence of CEC in tissue fragments used for MDM stimulation attenuated the upregulation of distinct pro-inflammatory chemokines, like MCP-3 and IL-8, reduced the monocyte survival time, and diminished monocyte-to-macrophage differentiation induced by conditioned media. Distinct anti-inflammatory cytokines, like IL-4 and IL-13, were upregulated in the presence of corneal endothelium. Cornea fragment-stimulated MDMs induced recruitment of monocytes from a PBMC pool in a transwell migration model, modulated immune cell viability and promoted further immune cell recruitment and differentiation.Human macrophages respond to allogenic corneal tissue and generate an inflammatory milieu. This can drive further recruitment of immunocompetent cells and modulate cell survival and differentiation of the cells recruited. These observations are consistent with the hypothesis that macrophages play a significant role in the initiation of corneal transplant rejection. Our data also indicate that distinct aspects of early human corneal transplant rejection can be modelled in vitro.
Project description:We previously reported that the stimulation of monocyte-derived macrophages (MDM) by plate-bound i.v. Igs inhibits HIV-1 replication. In this study, we show that IgG immune complexes also suppress HIV-1 replication in MDMs and that activating receptors for the Fc portion of IgG-FcgammaRI, FcgammaRIIA, and FcgammaRIII-are responsible for the inhibition. MDM stimulation through FcgammaRs induces activation signals and the secretion of HIV-1 modulatory cytokines, such as M-CSF, TNF-alpha, and macrophage-derived chemokine. However, none of these cytokines contribute to HIV-1 suppression. HIV-1 entry and postintegration steps of viral replication are not affected, whereas reduced levels of reverse transcription products and of integrated proviruses, as determined by real-time PCR analysis, account for the suppression of HIV-1 gene expression in FcgammaR-activated MDMs. We found that FcgammaR-dependent activation of MDMs also inhibits the replication of HIV-2, SIVmac, and SIVagm, suggesting a common control mechanism for primate immunodeficiency lentiviruses in activated macrophages.
Project description:BACKGROUND:Ischemic stroke provokes a neuroinflammatory response and simultaneously promotes release of epinephrine and norepinephrine by the sympathetic nervous system. This increased sympathetic outflow can act on ?2-adrenergic receptors expressed by immune cells such as brain-resident microglia and monocyte-derived macrophages (MDMs), but the effect on post-stroke neuroinflammation is unknown. Thus, we investigated how changes in ?2-adrenergic signaling after stroke onset influence the microglia/MDM stroke response, and the specific importance of microglia/MDM ?2-adrenergic receptors to post-stroke neuroinflammation. METHODS:To investigate the effects of ?2-adrenergic receptor manipulation on post-stroke neuroinflammation, we administered the ?2-adrenergic receptor agonist clenbuterol to mice 3?h after the onset of photothrombotic stroke. We immunostained to quantify microglia/MDM numbers and proliferation and to assess morphology and activation 3?days later. We assessed stroke outcomes by measuring infarct volume and functional motor recovery and analyzed gene expression levels of neuroinflammatory molecules. Finally, we evaluated changes in cytokine expression and microglia/MDM response in brains of mice with selective knockout of the ?2-adrenergic receptor from microglia and monocyte-lineage cells. RESULTS:We report that clenbuterol treatment after stroke onset causes enlarged microglia/MDMs and impairs their proliferation, resulting in reduced numbers of these cells in the peri-infarct cortex by 1.7-fold at 3?days after stroke. These changes in microglia/MDMs were associated with increased infarct volume in clenbuterol-treated animals. In mice that had the ?2-adrenergic receptor specifically knocked out of microglia/MDMs, there was no change in morphology or numbers of these cells after stroke. However, knockdown of ?2-adrenergic receptors in microglia and MDMs resulted in increased expression of TNF? and IL-10 in peri-infarct tissue, while stimulation of ?2-adrenergic receptors with clenbuterol had the opposite effect, suppressing TNF? and IL-10 expression. CONCLUSIONS:We identified ?2-adrenergic receptor signaling as an important regulator of the neuroimmune response after ischemic stroke. Increased ?2-adrenergic signaling after stroke onset generally suppressed the microglia/MDM response, reducing upregulation of both pro- and anti-inflammatory cytokines, and increasing stroke size. In contrast, diminished ?2-adrenergic signaling in microglia/MDMs augmented both pro- and anti-inflammatory cytokine expression after stroke. The ?2-adrenergic receptor may therefore present a therapeutic target for improving the post-stroke neuroinflammatory and repair process.
Project description:?7 nicotinic acetylcholine receptors (nAChRs) are ubiquitous in the nervous system and ensure important neurophysiological functionality for many processes. However, they are also found in cells of the immune system, where their role has been less studied. Here we report the pro-inflammatory effect of ImI, a well characterized conotoxin that inhibits ?7 nAChRs, on differentiated THP-1 pre-monocyte macrophages (MDM) obtained by phorbol 12-myristate 13 acetate (PMA) treatment. Enzyme-linked immunosorbent assay (ELISA) performed on supernatant fluids of LPS challenged MDM showed ImI-mediated upregulation of pro-inflammatory cytokine TNF-? in an ImI concentration-dependent manner from 0.5 to 5.0 µmol/L and for IL-8 up to 1.0 µmol/L. Levels of anti-inflammatory cytokine TGF-? remained practically unaffected in ImI treated MDMs. Nicotine at 10 µmol/L significantly downregulated the release of TNF-?, but showed a lesser effect on IL-8 secretion and no effect on TGF-?. Fluorescent competitive assays involving ImI, ?-bungarotoxin and nicotine using MDM and the murine macrophage RAW 264.7 suggest a common binding site in the ?7 receptor. This work extends the application of conotoxins as molecular probes to non-excitatory cells, such as macrophages and supports the involvement of the ?7 nAChR in regulating the inflammatory response via the cholinergic anti-inflammatory pathway (CAP).
Project description:Macrophages are a major target for human immunodeficiency virus type 1 (HIV-1) infection. However, macrophages are largely heterogeneous and may exhibit differences in permissiveness to HIV-1 infection. This study highlights the interplay of macrophage heterogeneity in HIV-1 pathogenesis. We show that monocyte-derived macrophages (MDMs) could be divided into two distinct subsets: CD14+Siglec-1hiCD4+ (non-adherent MDM) and CD14+Siglec-1LoCD4- (adherent MDM). The CD14+Siglec-1hiCD4+MDM subset represented the smaller proportion in the macrophage pool, and varied among different donors. Fractionation and subsequent exposure of the two MDM subsets to HIV-1 revealed opposite outcomes in terms of HIV-1 capture and infection. Although the CD14+Siglec-1hiCD4+MDM captured significantly more HIV-1, infection was significantly higher in the CD14+Siglec-1LoCD4-MDM subset. Thus, CD14+Siglec-1hiCD4+MDM were less permissive to infection. Depletion of CD14+Siglec-1hiCD4+MDM or a decrease in their percentage, resulted in increased infection of MDM, suggestive of a capacity of these cells to capture and sequester HIV-1 in an environment that hinders its infectivity. Increased expression of innate restriction factors and cytokine genes were observed in the non-adherent CD14+Siglec-1hiCD4+MDM, both before and after HIV-1 infection, compared to the adherent CD14+Siglec-1LoCD4-MDM. We speculate that the differential expression of gene expression profiles in the two macrophage subsets may provide an explanation for the differences observed in HIV-1 infectivity.
Project description:BACKGROUND:Galectin-3 is a 32?kDa protein secreted by macrophages involved in processes such as cell activation, chemotaxis and phagocytosis. Galectin-3 has previously been shown to improve the ability of airway macrophages to ingest apoptotic cells (efferocytosis) in chronic obstructive pulmonary disease (COPD) and may be of interest in non-eosinophilic asthma (NEA) which is also characterised by impaired efferocytosis. It was hypothesised that the addition of exogenous galectin-3 to monocyte-derived macrophages (MDMs) derived from donors with NEA would enhance their ability to engulf apoptotic granulocytes. METHODS:Eligible non-smoking adults with asthma (n?=?19), including 7 with NEA and healthy controls (n?=?10) underwent a clinical assessment, venepuncture and sputum induction. MDMs were co-cultured with apoptotic granulocytes isolated from healthy donors with or without exogenous recombinant galectin-3 (50??g/mL) and efferocytosis was assessed by flow cytometry. Galectin-3 expression and localisation in MDMs was visualised by immunofluorescence staining and fluorescence microscopy. Galectin-3, interleukin (IL)-6 and CXCL8 secretion were measured in cell culture supernatants by ELISA and cytometric bead array. RESULTS:Baseline efferocytosis (mean (±standard deviation)) was lower in participants with asthma (33.2 (±17.7)%) compared with healthy controls (45.3 (±15.9)%; p?=?0.081). Efferocytosis did not differ between the participants with eosinophilic asthma (EA) (31.4 (±19.2)%) and NEA (28.7 (±21.5)%; p?=?0.748). Addition of galectin-3 significantly improved efferocytosis in asthma, particularly in NEA (37.8 (±18.1)%) compared with baseline (30.4 (±19.7)%; p?=?0.012). Efferocytosis was not associated with any of the clinical outcomes but was negatively correlated with sputum macrophage numbers (Spearman r?=?-?0.671; p?=?0.017). Galectin-3 was diffusely distributed in most MDMs but formed punctate structures in 5% of MDMs. MDM galectin-3 secretion was lower in asthma (9.99 (2.67, 15.48) ng/mL) compared with the healthy controls (20.72 (11.28, 27.89) ng/mL; p?=?0.044) while IL-6 and CXCL8 levels were similar. CONCLUSIONS:Galectin-3 modulates macrophage function in asthma, indicating a potential role for galectin-3 to reverse impaired efferocytosis in NEA.