Macrophage Spreading Research Articles

Multiscale architecture design of 3D printed biodegradable Zn-based porous scaffolds for immunomodulatory osteogenesis

Reconciling the dilemma between rapid degradation and overdose toxicity is challenging in biodegradable materials when shifting from bulk to porous materials. Here, we achieve significant bone ingrowth into Zn-based porous scaffolds with 90% porosity via osteoinmunomodulation. At microscale, an alloy incorporating 0.8 wt% Li is employed to create a eutectoid lamellar structure featuring the LiZn4 and Zn phases. This microstructure optimally balances high strength with immunomodulation effects. At mesoscale, surface pattern with nanoscale roughness facilitates filopodia formation and macrophage spreading. At macroscale, the isotropic minimal surface G unit exhibits a proper degradation rate with more uniform feature compared to the anisotropic BCC unit. In vivo, the G scaffold demonstrates a heightened efficiency in promoting macrophage polarization toward an anti-inflammatory phenotype, subsequently leading to significantly elevated osteogenic markers, increased collagen deposition, and enhanced new bone formation. In vitro, transcriptomic analysis reveals the activation of JAK/STAT pathways in macrophages via up regulating the expression of Il-4, Il-10, subsequently promoting osteogenesis.

In vitro immunomodulatory effect of solid versus porous phosphate-based glass microspheres using macrophages

This study aimed to investigate the immunomodulatory effect of two different forms of phosphate-based glass microspheres (solid and porous), on human macrophages. Human THP-1 monocytes were converted to M0 macrophages after being treated with 100 ng/mL phorbol 12-myristate 13-acetate for 48 h. The differentiated cells were analysed for the CD14 marker using flow cytometry. The adhesion, spreading, and viability of M0 macrophages grown directly or indirectly (extracts) at varying concentrations of solid and porous glass microspheres (GMs) were analysed via phase contrast microscopy, confocal microscopy, and XTT assay. The expression of IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70 cytokines was investigated using flow cytometry. The conversion to M0 macrophages was confirmed by their adherent nature, increased granularity, and CD14 expression. The results showed that both solid and porous GMs or extracts favored the attachment, spreading, and proliferation of macrophages in a comparable manner to cells grown in a normal tissue culture medium. Only the higher concentration of porous GMs (10 mg/mL) changed the morphology of M0 macrophages and increased the expression of IL-1β and IL-8 pro-inflammatory cytokines; this could be related to the fast degradation nature of porous GMs. Of the six cytokines analysed, M0 macrophages grown directly or indirectly with GMs only expressed IL-1β, IL-10, and IL-8. Accordingly, solid microspheres may have advantages as regenerative agents due to their controlled degradation.

Toxicological Profile and Anti-Inflammatory Effect of Mucoadhesive Gel from Residues of Agave sisalana and Punica granatum.

Inflammation is a natural protective reaction of the body against endogenous and exogenous damage, such as tissue injuries, trauma, and infections. Thus, when the response is adequate, inflammation becomes a defense mechanism to repair damaged tissue, whereas when the response is inadequate and persistent, the increase in inflammatory cells, cytosines, and chymosins impair tissue regeneration and promote a response harmful to the organism. One example is chronic tissue inflammation, in which a simple lesion can progress to ulcers and even necrosis. In this situation, the anti-inflammatory medications available in therapy are not always effective. For this reason, the search for new treatments, developed from medicinal plants, has increased. In this direction, the plants Agave sisalana (sisal) and Punica granatum (pomegranate) are rich in saponins, which are secondary metabolites known for their therapeutic properties, including anti-inflammatory effects. Although Brazil is the world's leading sisal producer, approximately 95% of the leaves are discarded after fiber extraction. Similarly, pomegranate peel waste is abundant in Brazil. To address the need for safe and effective anti-inflammatory treatments, this study aimed to create a topical mucoadhesive gel containing a combination of sisal (RS) and pomegranate residue (PR) extracts. In vitro experiments examined isolated and combined extracts, as well as the resulting formulation, focusing on (1) a phytochemical analysis (total saponin content); (2) cytotoxicity (MTT assay); and (3) a pharmacological assessment of anti-inflammatory activity (phagocytosis, macrophage spreading, and membrane stability). The results revealed saponin concentrations in grams per 100 g of dry extract as follows: SR-29.91 ± 0.33, PR-15.83 ± 0.93, association (A)-22.99 ± 0.01, base gel (G1)-0.00 ± 0.00, and association gel (G2)-0.52 ± 0.05. In MTT tests for isolated extracts, cytotoxicity values (µg/mL) were 3757.00 for SR and 2064.91 for PR. Conversely, A and G2 exhibited no cytotoxicity, with increased cell viability over time. All three anti-inflammatory tests confirmed the presence of this activity in SR, PR, and A. Notably, G2 demonstrated an anti-inflammatory effect comparable to dexamethasone. In conclusion, the gel containing SR and PR (i.e., A) holds promise as a novel herbal anti-inflammatory treatment. Its development could yield economic, social, and environmental benefits by utilizing discarded materials in Brazil.

Deletion of the gene for the African swine fever virus BCL-2 family member A179L increases virus uptake and apoptosis but decreases virus spread in macrophages and reduces virulence in pigs.

African swine fever virus (ASFV) causes a lethal disease of pigs with high economic impact in affected countries in Africa, Europe, and Asia. The virus encodes proteins that inhibit host antiviral defenses, including the type I interferon response. Host cells also activate cell death through a process called apoptosis to limit virus replication. We showed that the ASFV A179L protein, a BCL-2 family apoptosis inhibitor, is important in reducing apoptosis in infected cells since deletion of this gene increased cell death and reduced virus replication in cells infected with the A179L gene-deleted virus. Pigs immunized with the BeninΔA179L virus showed no clinical signs and a weak immune response but were not protected from infection with the deadly parental virus. The results show an important role for the A179L protein in virus replication in macrophages and virulence in pigs and suggest manipulation of apoptosis as a possible route to control infection.

The CIS association of CD47 with integrin Mac-1 regulates macrophage responses by stabilizing the extended integrin conformation

CD47 is a ubiquitously expressed cell surface integrin-associated protein. Recently, we have demonstrated that integrin Mac-1 (αMβ2, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, can be coprecipitated with CD47. However, the molecular basis for the CD47-Mac-1 interaction and its functional consequences remain unclear. Here, we demonstrated that CD47 regulates macrophage functions directly interacting with Mac-1. In particular, adhesion, spreading, migration, phagocytosis, and fusion of CD47-deficient macrophages were significantly decreased. We validated the functional link between CD47 and Mac-1 by co-immunoprecipitation analysis using various Mac-1-expressing cells. In HEK293 cells expressing individual αM and β2 integrin subunits, CD47 was found to bind both subunits. Interestingly, a higher amount of CD47 was recovered with the free β2 subunit than in the complex with the whole integrin. Furthermore, activating Mac-1-expressing HEK293 cells with PMA, Mn2+, and activating antibody MEM48 increased the amount of CD47 in complex with Mac-1, suggesting CD47 has a greater affinity for the extended integrin conformation. Notably, on the surface of cells lacking CD47, fewer Mac-1 molecules could convert into an extended conformation in response to activation. Additionally, we identified the binding site in CD47 for Mac-1 in its constituent IgV domain. The complementary binding sites for CD47 in Mac-1 were localized in integrin epidermal growth factor-like domains 3 and 4 of the β2 and calf-1 and calf-2 domains of the α subunits. These results indicate that Mac-1 forms a lateral complex with CD47, which regulates essential macrophage functions by stabilizing the extended integrin conformation.

Differential nuclear translocation of STAT6 regulates the mechanosensitive immune cell activation.

Alternatively activated or M2 macrophages, as opposed to the well characterized pro-inflammatory or M1 macrophages, vitally regulate anti-inflammation, wound healing, and tissue repair to maintain tissue homeostasis. Although ubiquitous presence of macrophages in diverse tissues, exposed to different physical environments, infers distinct immune responses of M2 macrophages with high phenotypic heterogeneity, the underlying mechanism of how the varying extracellular mechanical conditions alter their immunological activation remains unclear. Here, we demonstrate that M2 activation requires a threshold mechanical cue from the extracellular microenvironment, and matrix rigidity-dependent macrophage spreading is mediated by the F-actin formation that is essential to regulate mechanosensitive M2 activation of macrophages. We identified a new mechanosensing function of STAT6 (signal transducer and activator of transcription 6), a key transcription factor for M2 activation, whose intranuclear transportation is promoted by the rigid matrix that facilitates the F-actin formation. Our findings further highlight the critical role of mechanosensitive M2 activation of macrophages in long-term adaptation to the extracellular microenvironment by bridging nuclear mechanosensation and immune responses.

Bridging the Gap: Exploring the Preclinical Potential of Pereskia grandifolia in Metabolic-Associated Fatty Liver Disease.

Metabolic-associated fatty liver disease (MAFLD) is a complex condition characterized by steatosis and metabolic disturbances. Risk factors such as diabetes, cigarette smoking, and dyslipidaemia contribute to its development and progression. Effective and safe therapies for MAFLD are urgently needed. Pereskia grandifolia has shown potential as an alternative treatment, but its effectiveness against liver disease remains unexplored. This research aims to determine the hepatoprotective properties of P. grandifolia using a model of MAFLD. The study was carried out through various phases to assess the safety and efficacy of the ethanol-soluble fraction of P. grandifolia. Initially, an in vitro assay was performed to assess cell viability. This was followed by an acute toxicity test conducted in rats to determine the safety profile of the extract. Subsequently, the anti-inflammatory properties of P. grandifolia were examined in macrophages. For the MAFLD study, diabetic Wistar rats were made diabetic and exposed to a high fat diet and cigarette smoke, for 4 weeks. During the last 2 weeks, the rats were orally given either the vehicle (negative control group; C-), P. grandifolia (30, 100, and 300 mg/kg), or insulin in addition to simvastatin. A basal group of rats not exposed to these risk factors was also assessed. Blood samples were collected to measure cholesterol, triglycerides, glucose, ALT, and AST levels. Liver was assessed for lipid and oxidative markers, and liver histopathology was examined. P. grandifolia showed no signs of toxicity. It demonstrated anti-inflammatory effects by inhibiting phagocytosis and macrophage spreading. The MAFLD model induced liver abnormalities, including increased AST, ALT, disrupted lipid profile, oxidative stress, and significant hepatic damage. However, P. grandifolia effectively reversed these changes, highlighting its potential as a therapeutic agent. These findings emphasize the significance of P. grandifolia in mitigating hepatic consequences associated with various risk factors.

Nuclear transport of STAT6 determines the matrix rigidity dependent M2 activation of macrophages

Alternatively activated or M2 macrophages, as opposed to the well characterized pro-inflammatory or M1 macrophages, vitally regulate anti-inflammation, wound healing, and tissue repair to maintain tissue homeostasis. Although ubiquitous presence of macrophages in diverse tissues, exposed to different physical environments, infers distinct immune responses of M2 macrophages with high phenotypic heterogeneity, the underlying mechanism of how the varying extracellular mechanical conditions alter their immunological activation remains unclear. Here, we demonstrate that M2 activation requires a threshold mechanical cue from the extracellular microenvironment, and matrix rigidity-dependent macrophage spreading is mediated by the F-actin formation that is essential to regulate mechanosensitive M2 activation of macrophages. We identified a new mechanosensing function of STAT6 (signal transducer and activator of transcription 6), a key transcription factor for M2 activation, whose intranuclear transportation is promoted by the rigid matrix that facilitates the F-actin formation. Our findings further highlight the critical role of mechanosensitive M2 activation of macrophages in long-term adaptation to the extracellular microenvironment by bridging nuclear mechanosensation and immune responses.

A Novel Class of HIV-1 Inhibitors Targeting the Vpr-Induced G2-Arrest in Macrophages by New Yeast- and Cell-Based High-Throughput Screening.

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, arrests the cell cycle of the G2 phase, and this Vpr-mediated G2 arrest is implicated in an efficient HIV-1 spread in monocyte-derived macrophages. Here, we screened new candidates for Vpr-targeting HIV-1 inhibitors by using fission yeast- and mammalian cell-based high-throughput screening. First, fission yeast strains expressing the HIV-1 Vpr protein were generated and then treated for 48 h with 20 μM of a synthetic library, including 140,000 chemical compounds. We identified 268 compounds that recovered the growth of Vpr-overexpressing yeast. The selected compounds were then tested in mammalian cells, and those displaying high cytotoxicity were excluded from further cell cycle analysis and imaging-based screening. A flow cytometry analysis confirmed that seven compounds recovered from the Vpr-induced G2 arrest. The cell toxicity and inhibitory effect of HIV-1 replication in human monocyte-derived macrophages (MDM) were examined, and three independent structural compounds, VTD227, VTD232, and VTD263, were able to inhibit HIV-1 replication in MDM. Furthermore, we showed that VTD227, but not VTD232 and VTD263, can directly bind to Vpr. Our results indicate that three new compounds and their derivatives represent new drugs targeting HIV-1 replication and can be potentially used in clinics to improve the current antiretroviral therapy.

Macrophage activity regulation by high dilution of aspirin and its possible mechanism

Background: Under LPS-stimulus, platelets can activate macrophages by a cell-to-cell contact or through cytokine degranulation. Rebound effects of anti-thrombotic agents, such as prostanoids and COX inhibitors can lead to thrombosis, infarct, and stroke. Aspirin has been prescribed for decades due to its powerful antiplatelet action, but it is also related to paradoxical effects such as withdrawal syndrome peaks, resistance, and thrombogenesis. Ultra-diluted aspirin can also produce the same effect in one hour, regardless of Cox-2, by still unknown pathways. Antithrombotic effects of aspirin are also reversed by its high dilutions. Aims: This study aims to characterize the effects of aspirin 15cH on macrophages challenged with LPS, a Cox-2 activator. Methodology: RAW 264.7 macrophages were sown in 24 wells plates using R10 medium, boosted with 1μg/ml LPS, and treated with aspirin 15 cH and controls. The activity was evaluated after 24 hours. Supernatants were evaluated for cytokines, nitric oxide, and dielectric oscillations, through solvatochromic dyes (Cartwright’s method). Results and discussion: macrophage spreading was increased by aspirin 15 cH, an LPS-like effect. Paradoxically, a significant reduction of this effect was noted when both, LPS and aspirin 15 cH, were added. Succussed water reversed the effect of LPS, leading to TNF (p<0.05) production close to baseline levels. Also, the single treatment with succussed water inhibited IL-10 production (p<0.05), but aspirin 200 µg/mL (positive control) highly increased it (p<0.0001), showing the validity of the model. Nitric oxide production was strengthened by LPS presence (p<0.0001), as expected, but partially downregulated after treatment with aspirin 200 µg/mL, water and succussed water. A pilot study with solvatochromic dyes showed no significant difference among treatments. Conclusion: The main data suggest that aspirin 15 cH increases macrophage activity but presents a paradoxal effect when mixed with LPS. On the other hand, succussed water itself has modulatory effects on macrophages.

Interaction of the Class A Scavenger Receptor with Multiple Myeloma Cells

Tumor‐associated macrophages (TAMs) play a central role in regulating the immune response within the cancer microenvironment. TAMs express Class A Scavenger Receptors (SR‐A), which are pattern recognition receptors that bind a wide array of ligands. SR‐A expression by TAMs is often associated with an immune‐suppressive macrophage phenotype. Multiple myeloma (MM) is a malignant disease of the bone marrow characterized by the proliferation of differentiated plasma cells. The disease progresses from asymptomatic monoclonal gammopathy of undetermined significance (MGUS) to smoldering myeloma (SMM) and finally to symptomatic MM. In many solid tumors, the number of SR‐A‐expressing TAMs correlates with tumor aggressiveness and poor prognosis. Although SR‐A‐expressing macrophages are abundant in the bone marrow, the importance of SR‐A in the progression of MM is unknown. All patient samples for the study were collected under IRB approved protocol 260665. To determine if SR‐A may be important in MM, we utilized a multiplexed immunofluorescence (IF) approach to localize SR‐A‐expressing macrophages within the MM bone marrow microenvironment. Macrophages were detected using CD163 and SR‐A antibodies and plasma cells were identified using CD138 antibody. Multiplexed‐IF staining showed that SR‐A‐positive macrophages were localized near MM cells. This was especially evident in tumor‐rich regions where SR‐A‐expressing TAMs displayed a distinct branched morphology. SR‐A binding to ligand induces macrophage attachment and spreading. Thus, we hypothesized that the localization of SR‐A‐expressing TAMs near MM cells results from SR‐A binding to a ligand expressed on these cells. We tested this possibility using a flow cytometry‐based assay to measure the binding of a soluble SR‐A (sSR‐A) protein with MM cell lines and plasma cells obtained from bone marrow aspirates of MM patients. sSR‐A was able to bind to all six different MM cell lines that were examined. In bone marrow aspirates (n=15), sSR‐A bound to neoplastic plasma cells (CD138+CD45‐) but not to non‐plasma cells (CD45+). sSR‐A binding to neoplastic plasma cells was confirmed using enriched plasma cells isolated from bone marrow aspirates using anti‐CD138 magnetic beads. sSR‐A binding to plasma cells was abolished by the SR‐A ligand, fucoidan, demonstrating specificity. Together, our results demonstrate that SR‐A is expressed on TAMs within the MM microenvironment and binds to MM cells. SR‐A binding to MM cells may be a mechanism for regulating TAM localization and function in the tumor microenvironment.

Characterization of Macrophage and Cytokine Interactions with Biomaterials Used in Negative-Pressure Wound Therapy.

Macrophages are innate immune cells that help wounds heal. Here, we study the potential immunomodulatory effects of negative-pressure wound therapy (NPWT) materials on the macrophage inflammatory response. We compared the effects of two materials, Granufoam™ (GF) and Veraflo Cleanse™ (VC), on macrophage function in vitro. We find that both materials cause reduced expression of inflammatory genes, such as TNF and IL1B, in human macrophages stimulated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFNγ). Relative to adherent glass control surfaces, VC discourages macrophage adhesion and spreading, and may potentially sequester LPS/IFNγ and cytokines that the cells produce. GF, on the other hand, was less suppressive of inflammation, supported macrophage adhesion and spreading better than VC, and sequestered lesser quantities of LPS/IFNγ in comparison to VC. The control dressing material cotton gauze (CT) was also immunosuppressive, capable of TNF-α retention and LPS/IFNγ sequestration. Our findings suggest that NPWT material interactions with cells, as well as soluble factors including cytokines and LPS, can modulate the immune response, independent of vacuum application. We have also established methodological strategies for studying NPWT materials and reveal the potential utility of cell-based in vitro studies for elucidating biological effects of NPWT materials.

TBC1D10C is a cytoskeletal functional linker that modulates cell spreading and phagocytosis in macrophages

Cell spreading and phagocytosis are notably regulated by small GTPases and GAP proteins. TBC1D10C is a dual inhibitory protein with GAP activity. In immune cells, TBC1D10C is one of the elements regulating lymphocyte activation. However, its specific role in macrophages remains unknown. Here, we show that TBC1D10C engages in functions dependent on the cytoskeleton and plasma membrane reorganization. Using ex vivo and in vitro assays, we found that elimination and overexpression of TBC1D10C modified the cytoskeletal architecture of macrophages by decreasing and increasing the spreading ability of these cells, respectively. In addition, TBC1D10C overexpression contributed to higher phagocytic activity against Burkholderia cenocepacia and to increased cell membrane tension. Furthermore, by performing in vitro and in silico analyses, we identified 27 TBC1D10C-interacting proteins, some of which were functionally classified as protein complexes involved in cytoskeletal dynamics. Interestingly, we identified one unreported TBC1D10C-intrinsically disordered region (IDR) with biological potential at the cytoskeleton level. Our results demonstrate that TBC1D10C shapes macrophage activity by inducing reorganization of the cytoskeleton-plasma membrane in cell spreading and phagocytosis. We anticipate our results will be the basis for further studies focused on TBC1D10C. For example, the specific molecular mechanism in Burkholderia cenocepacia phagocytosis and functional analysis of TBC1D10C-IDR are needed to further understand its role in health and disease.

In vitro study of homeopathic medicines in macrophages co-cultured with Leishmania (L.) amazonensis

Background: In previous studies the effect of homeopathic medicines in murine cutaneous Leishmaniasis were described, indicating immunomodulation. The phagocytes migration and their activity increased after Thymulin 5cH and decreased after Antimonium crudum 30cH treatment. 
 Methods: The morpho-functional features of RAW macrophages co-cultured with Leishmania (L.) amazonensis in RPMI medium were analyzed in vitro, after 24h treatment with 20% of Thymulin 5cH, 6cH and 7cH or Antimonium crudum 6cH, 30cH and 200cH. All assays were performed in blind and in three series of quadruplicate. The spreading was analyzed by the breadth and area of each macrophage, photographed in a NIKON Eclipse 200-Coolpix system and measured by Metamorph® Image analysis software. The phagocytosis was analyzed by the percentage of amastigotes incorporated into the phagocyte vacuoles. ANOVA and Tuckey Krammer were used as statistical methods. 
 Results: A marked increase of macrophage spreading (considering breadth and area) was seen in Thymulin 5cH and Thymulin 7cH treated cells (p≤0.01), as in Antimonium crudum 30cH (p=0.05) and 200cH (p=0.001) treated cells. Only Antimonium crudum 200cH presented increase in phagocytosis index (p=0.001). 
 Conclusions: The in vitro assay corroborates the findings obtained from in vivo studies and represents the first step to a deeper understanding of the mechanism of action.

Macrophage Phenotypic Changes on FN-Coated Physical Gradient Hydrogels.

The chemical and physical properties are two crucial cues when designing tissue engineering scaffold to mimic living tissue. Macrophages, the major players in the immune response, react rapidly to microenvironmental signals, including gradients of physical or chemical cues. Spatiotemporal gradients can modulate cell behavior, such as polarization, proliferation, and adhesion. Here, we studied macrophage phenotypic changes on untreated and fibronectin (FN)-coated methacrylated gellan gum with varying stiffnesses. The compressive moduli of hydrogel with different stiffnesses ranged from ∼5 to 30 kPa. Fibronectin was chemically attached to the substrate to facilitate macrophage proliferation, adhesion, and polarization. Classically (M1) and alternatively (M2) activated macrophages were cultured on both untreated and FN-coated gels. FN-coated substrates elevated cell numbers and enhanced macrophage spreading. The urea/nitrite ratio indicated that untreated rigid substrates shifted both polarizations toward a more proinflammatory phenotype. FN-coated substrates had no impact on M1 polarization. In contrast, FN-coated stiffer gels polarized M2 cells toward an anti-proinflammatory state based on arginine activity and CD206 expression. In addition, macrophage polarization on the softer gel was not influenced by the neighboring cells cultured on the stiffer side of the gel. Using mechanical gradients to control macrophage polarization can be a useful tool in ensuring a proper healing response and for tissue engineering.

Comparison of effects of homeopathic medicines prepared in glass and plastic vials in macrophage activity in vitro

Background: According to the “silica hypothesis” to explain the action of homeopathic medicines, complex nanoparticles involving the silica extracted from the walls of the glass vial during succussions would be needed to act on target cells. Aims: We aimed to know if suspended particles of silica found in Arsenicum album (AA) could be associated to its biological effects in vitro. Methodology: RAW 264.7 macrophages were co-cultured with yeast (Saccharomyces cerevisiae) to promote phagocytosis. During this process, the cells were exposed to AA in different homeopathic dilutions (30K, 6cH and 200cH) prepared in both, glass or silica-fee plastic vials. All dilutions were prepared from the same 3cH matrix. Since parallel experiments showed random stimulating and inhibiting effects of homeopathic lactose, untreated cells and water were used as controls. The macrophage activity was measured by digital histometry (Metamorph®), reflecting the cell spreading and phagocytosis. A complementary analysis using the acridine orange stain in real time fluorescence microscopy was also performed to evaluate the organization of phago-lysosomes inside the cells. All experiments were performed in triplicate. Microparticles present in the medicines were identified in an EDS (energy dispersive x-ray detector) system (JEOL JMS 6510), according to their chemical elements composition. The morphology of particles was not considered for analysis, since it was considered irrelevant. Results: Experiment one: AA 30K and pure water, when prepared in plastic vials, were able to increase macrophage activity (p≤0.05), but the same result was not seen in cells treated with AA 30K or pure water stocked in glass. Experiment two: AA 200cH prepared in plastic vials also lead to significant increase of macrophage spreading (p≤0.05) in relation to the controls, but no effect was seen in the samples prepared in glass vials. Experiment three: No difference was observed in AA 6cH treated cells in relation to the controls. The morphology of the cells under fluorescence microscopy exhibited increase in lysosome activity in AA 200cH prepared in both plastic and glass vials, in relation to the untreated control. The EDS analysis has shown that only medicines prepared in glass vials contained suspended silica particles. Conclusions: Taken the data together, the unspecific effects on macrophage spreading after treatment with homeopathic AA 30K, 200cH and water prepared in plastic vials, added to the absence of silica particles in these preparations and the increase of lysosomal activity only seen in AA 200cH, independently of the vial nature, corroborate that silica might not be a mandatory condition to justify the biological effects of homeopathic medicines. Additionally, the existence of putative contaminants released from plastic vials during the medicine manipulation deserves further studies.
 Acknowledgements: CAPES, UNIP, ABFH

Effects of Antimomium crudum 30cH and 200cH on the macrophage – Leishmania (L) amazonensis relation in vitro

Background: Leishmaniasis is a chronic skin and systemic disease, whose treatment is related to important side effects and loss of life quality. In previous results [1], mice treated with Antimonium crudum (AC) 30cH presented reduction in local inflammatory process and modulation of B1 cell-phagocyte differentiation and migration, but also increase of free amastigotes number among the surrounding tissue. Aims: To know the mechanisms involved, a series of in vitro studies was done, using co-cultures of macrophages (RAW 264.7) and Leishmania (L.) amazonensis treated with AC 30cH and 200cH, in different times. Methodology: The morpho-functional features of macrophages (spreading, phagocytosis and oxidative activity), the number of free promastigotes in the supernatant and the cytokines measurement were evaluated. The spreading and phagocytosis assays were performed in quadruplicate, in three experiments, resulting in 12 datapoints for each dilution/time. Kruskal Wallis (for no parametric variables) and two-way ANOVA test (for parametric variables) were used to verify time to time differences and the interaction between treatment and time respectively, being p≤0.05 considered significant. Animals were not used in this study. Results: The treatment with AC 30cH and AC 200cH resulted in significant but transitory increase in spreading and phagocytosis activity after 2 to 24 hours of incubation, followed by increase in the number of free promastigotes in the supernatant (in AC 30cH treatment) and decrease in CD86 expression (in AC 200cH treatment). GMCSF, alpha-INF, IL1, IL6, IL10 and IL12 were reduced after 48 to 72 hours and CCL4 and RANTES were reduced after 120H, for both potencies. Two peaks of CCL2 were seen in Leishmania sp infected macrophages, at 24 and 120 hours, but only AC30cH inhibited them. A peak of VEGF was observed after 120 hours following the treatment with AC 200cH, together with the increase in the number of multinucleated cells. The morphology of macrophages at fluorescence microscopy after the staining with acridine orange in fresh unfixed cells showed severe reduction of acid vacuoles in AC 30CH treated cells at 2 hours of parasite-macrophage interaction, revealing possible macrophage anergy. No difference in peroxide / NO production and in apoptosis percentage of free promastigotes was seen among groups, in all evaluated times. Conclusions: Both potencies were able to decrease most of cytokines production, specially CCL2 in AC 30cH treated cells, which explains the inhibition of monocyte migration seen in vivo [1]. The late peak of VEGF observed in AC 200cH treated cells suggests a M1 - M2 polarization, whose biological meaning is still under discussion.
 
 Acknowledgements: CAPES, UNIP, FAPESP (2014/00967-1)
  
 References
 Rodrigues de Santana F, de Paula Coelho C, Cardoso TN, Perez Hurtado EC, Roberti Benites N, Dalastra Laurenti M, Villano Bonamin L. Modulation of inflammation response to murine cutaneous Leishmaniasis by homeopathic medicines: Antimonium crudum 30cH. Homeopathy, 2014;103(4):264-74. doi: 10.1016/j.homp.2014.08.006.

Searching for the mechanisms involved in Antimonium crudum action on macrophage – Leishmania interaction in vitro

Searching for the mechanisms involved in Antimonium crudum action on macrophage – Leishmania interaction in vitro

The Cis Interaction of CD47 with Integrin Mac‐1 (α M β 2 , CD11b/CD18) Regulates Macrophage Responses

CD47 is a widely expressed cell surface receptor that has originally been identified as an integrin-associated protein. In immune cells, CD47 has been shown to associate in cis with several integrins of the β1 and β3 subfamilies and the one member of the β2 subfamily, αLβ2. Surprisingly, the association of CD47 with integrin Mac-1 (αMβ2, CD11b/CD18), the major adhesion receptor on the surface of myeloid cells has not been documented. Furthermore, while previous studies focused on the mechanism by which CD47 on various host cells prevents phagocytosis by macrophages, the question as to how CD47 on macrophages may influence the responses of these cells has not been addressed. Here, we demonstrated that CD47 modulates Mac-1-dependent macrophage functions. In particular, adhesion of macrophages isolated from CD47-/- mice to fibrinogen and ICAM-1, the established physiological ligands of Mac-1 was significantly decreased compared to wild-type counterparts. Also, spreading of CD47-deficient macrophages was decreased by four- and two-fold on fibrinogen and ICAM-1, respectively. Compared to wild-type macrophages, migration of CD47-deficient macrophages to the Mac-1 ligand, the cathelicidin peptide LL-37 was significantly reduced. The lack of CD47 on the surface of macrophages impaired their ability to fuse in the presence of IL-4. Finally, the deficiency of CD47 also reduced phagocytosis of opsonized latex beads, a process fully dependent on Mac-1. The functional link between CD47 and Mac-1 was confirmed by their physical association in cis on the surface of macrophages and Mac-1-expressing HEK293 cells using co-immunoprecipitation and the proximity ligation assay. Studies with HEK293 ceIls expressing individual αM or β2 integrin subunits of Mac-1 showed that both subunits can interact with CD47. The activation of cells with PMA, Mn2+, and mAb MEM48 increased the amount of CD47 recovered in immune complexes with Mac-1, suggesting that CD47 has increased affinity for integrin in the extended conformation. The binding site in CD47 for Mac-1 was identified in its constituent IgV domain as recombinant IgV blocked the Mac-1-CD47 complex formation and inhibited adhesion and phagocytosis. Collectively, these results indicate that Mac-1 forms a lateral complex with CD47, which regulates important macrophage functions.

CD47 Ligation Repositions the Inhibitory Receptor SIRPA to Suppress Integrin Activation and Phagocytosis

CD47 acts as a "don't eat me" signal that protects cells from phagocytosis by binding and activating its receptor SIPRA on macrophages. CD47 suppresses multiple different pro-engulfment "eat me" signals, including immunoglobulin G (IgG), complement, and calreticulin, on distinct target cells. This complexity has limited understanding of how the "don't eat me" signal is transduced biochemically. Here, we utilized a reconstituted system with a defined set of signals to interrogate the mechanism of SIRPA activation and its downstream targets. CD47 ligation altered SIRPA localization, positioning SIRPA for activation at the phagocytic synapse. At the phagocytic synapse, SIRPA inhibited integrin activation to limit macrophage spreading across the surface of the engulfment target. Chemical reactivation of integrin bypassed CD47-mediated inhibition and rescued engulfment, similar to the effect of a CD47 function-blocking antibody. Thus, the CD47-SIRPA axis suppresses phagocytosis by inhibiting inside-out activation of integrin signaling in the macrophage, with implications to cancer immunotherapy applications.