Phagocytosis Of Latex Beads Research Articles

Glucomannan is a promising isoniazid's enhancer that inducing macrophage phagocytosis.

Isoniazid (INH) is a frontline antituberculosis agent effective against Mycobacterium tuberculosis (Mtb), but the increasing challenge of avoiding multidrug-resistant tuberculosis, including INH resistance, necessitates innovative approaches. This study focused on enhancing macrophage phagocytosis to overcome INH resistance. Glucomannan, an immunomodulatory polysaccharide, emerged as a potential macrophage activator. Our objective was to characterize the glucomannan-INH mixture and assess its impact on INH efficacy and macrophage activity. Detailed examination of the glucomannan from Amorphophallus muelleri (0.05%-0.2%) was performed in several methods. INH sensitivity tests were carried out with the Mtb strain H37RV on Löwenstein-Jensen medium. Murine macrophage (RAW264.7) viability and activity were evaluated through MTT and latex bead phagocytosis assays. Ultraviolet-wavelength spectrophotometry was used to analyze chemical structure changes. Glucomannan (0.05%-0.2%) significantly enhanced murine macrophage viability and activity. When glucomannan was combined with INH, the IC50 value was greater compared to INH only. Phagocytosis assays revealed heightened macrophage activity in the presence of 0.05% and 0.1% glucomannan. Importantly, glucomannan did not compromise INH efficacy or alter its chemical structure. This study underscores the potential of glucomannan, particularly with a lower molecular weight, as a promising enhancer of INH, boosting macrophage phagocytosis against INH-resistant Mtb. These findings challenge the assumptions about the impact of glucomannan on drug absorption and prompt potential reevaluation. While specific receptors for glucomannan in macrophage phagocytosis require further exploration, the complement receptors are proposed to be potential mediators.

Gelatin but not type I collagen promotes bacteria phagocytosis in PMA-treated U937 human lymphoma cells

ABSTRACT Purpose Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells. Materials and Methods Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted. Results Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis. Conclusion This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy.

Treponema pallidum protein Tp47 induced prostaglandin E2 to inhibit the phagocytosis in human macrophages.

During Treponema pallidum (T. pallidum) infection, the host's immune system actively engages in pursuit and elimination of T. pallidum, while T. pallidum skillfully employs various mechanisms to evade immune recognition. Macrophages exhibit incomplete clearance of T. pallidum invitro and the underlying mechanism of how T. pallidum resists the attack of macrophage remains unclear. To investigate the effect of T. pallidum membrane protein Tp47 on the phagocytosis of macrophages. THP-1-derived macrophages were used to investigate the role of Tp47 in the secretion of Prostaglandin E2 (PGE2) in macrophages and the mechanism by which Tp47 induced the production of PGE2, as well as the impact of PGE2 on the macrophage's phagocytosis. Tp47 (1-10 μg/mL) significantly inhibited the phagocytosis of latex beads and T. pallidum in macrophages (p ≤ 0.05). PGE2 production by macrophages could be induced by Tp47, and the phagocytic function of macrophages could be restored using PGE2 antibody. Tp47 produced PGE2 by activating the PERK/NF-κB/COX-2 pathway in macrophages. Inhibitors targeting PERK, NF-κB and COX-2, respectively, reduced the level of PGE2 and restored the phagocytic function of macrophages. Tp47-induced PGE2 production via the PERK/NF-κB/COX-2 pathway contributed to macrophage phagocytosis inhibition, which potentially contributes to immune evasion during the T. pallidum infection.

Characterization of polarization states of canine monocyte derived macrophages.

Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune‑related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.

Effects of exposure to alternating low-intensity, intermediate-frequency electric fields on the differentiation of human leukemic cell line U937.

Studying the bioeffects of electric fields have been the subject of ongoing research which led to promising therapeutic effect, particularly in cancer treatment. Here, we investigated the impact of low-intensity, intermediate-frequency alternating electric fields on the differentiation of human myeloid leukemia cell line U937. The results showed a near twofold increase in differentiation of U937 cells treated for 24 h by alternating 600 kHz, 150 V/m electric fields, in comparison to their control groups. This measure was evaluated by latex bead phagocytosis assay, nitro blue tetrazolium test, and cell cycle analysis which revealed a significant shift in the number of cells from G2 +M to G0 +G1 phases. The simulation result for the intracellular field intensity showed around 50% attenuation with respect to the applied external field for our setup which ruled out masking of the applied field by the internal electric noise of the cell. Based on previous studies we postulate a possible calcium-related effect for the observed differentiation, yet the exact underlying mechanism requires further investigation. Finally, our results may offer a potential therapeutic method for leukemia in the future.

Effect of taxifolin, a conjugate of taxifolin with glyoxylic acid, and naringenin on the functional activity of neutrophils

The aim of the present work was to study the effects of taxifolin, a conjugate of taxifolin with glyoxylic acid, and naringenin on phagocytosis of latex beads by neutrophils and adhesive properties of these cells. It was shown that taxifolin had no effect on the phagocytic activity and adhesive properties of neutrophils. A conjugate of taxifolin with glyoxylic acid strongly inhibited the phagocytic activity of neutrophils. At the same time, the conjugate significantly enhanced adhesion of these cells. Naringenin decreased phagocytic activity of neutrophils, though to a lesser extent than the conjugate did. Naringenin also inhibited adhesion of neutrophils. Thus, the data collected during our study indicate that polyphenols may influence the functional activity of neutrophils that can play an important role in the modulation of inflammation, especially when prolonged activation of neutrophils leads to tissue damage.

Atg2 Regulates Cellular and Humoral Immunity in Drosophila.

Autophagy is a process that promotes the lysosomal degradation of cytoplasmic proteins and is highly conserved in eukaryotic organisms. Autophagy maintains homeostasis in organisms and regulates multiple developmental processes, and autophagy disruption is related to human diseases. However, the functional roles of autophagy in mediating innate immune responses are largely unknown. In this study, we sought to understand how Atg2, an autophagy-related gene, functions in the innate immunity of Drosophila melanogaster. The results showed that a large number of melanotic nodules were produced upon inhibition of Atg2. In addition, inhibiting Atg2 suppressed the phagocytosis of latex beads, Staphylococcus aureus and Escherichia coli; the proportion of Nimrod C1 (one of the phagocytosis receptors)-positive hemocytes also decreased. Moreover, inhibiting Atg2 altered actin cytoskeleton patterns, showing longer filopodia but with decreased numbers of filopodia. The expression of AMP-encoding genes was altered by inhibiting Atg2. Drosomycin was upregulated, and the transcript levels of Attacin-A, Diptericin and Metchnikowin were decreased. Finally, the above alterations caused by the inhibition of Atg2 prevented flies from resisting invading pathogens, showing that flies with low expression of Atg2 were highly susceptible to Staphylococcus aureus and Erwinia carotovora carotovora 15 infections. In conclusion, Atg2 regulated both cellular and humoral innate immunity in Drosophila. We have identified Atg2 as a crucial regulator in mediating the homeostasis of immunity, which further established the interactions between autophagy and innate immunity.

Regulation of the phagocytic activity of astrocytes by neuroimmune mediators endogenous to the central nervous system.

The phagocytic activity of glial cells is essential for maintaining normal brain activity, and its dysfunction may contribute to the central nervous system (CNS) pathologies, including neurodegenerative diseases. Phagocytic activity is one of the well-established neuroimmune functions of microglia. Although emerging evidence indicates that astrocytes can also function as CNS phagocytes in humans and rodents, limited information is available about the molecular mechanism regulating this function. To address this knowledge gap, we studied modulation of the phagocytic activity of human U118 MG astrocytic cells and murine primary astrocytes by four CNS inflammatory mediators and bacterial endotoxin lipopolysaccharide (LPS). LPS and cytochrome c (CytC) upregulated, while interferon (IFN)-γ downregulated, phagocytosis of latex beads by human astrocytic cells and phagocytosis of synaptosomes by murine primary astrocytes. Interleukin (IL)-1β and tumor necrosis factor (TNF)-α had no effect on the phagocytic activity of human astrocytic cells but upregulated this function in murine astrocytes. Varying effects of combinations of the above inflammatory mediators were observed in these two cell types. LPS- and CytC-induced phagocytic activity of human astrocytic cells was partially mediated by activation of toll-like receptor 4 (TLR4). By monitoring other functions of astrocytes, we concluded there were no correlations between the effects of the mediators studied on astrocyte phagocytic activity and their secretion of cytokines, cytotoxins, or glutamate. Our study identified four candidate CNS regulators of astrocyte phagocytic activity. Future investigation of molecular mechanisms behind this regulation could identify novel therapeutic targets allowing modulation of this astrocyte-mediated clearance mechanism in CNS pathologies.

Beclin 1 regulates astrocyte phagocytosis and phagosomal recruitment of retromer

Phagocytosis plays an important role in maintaining brain homeostasis and when impaired can result in the accumulation of unwanted cellular material. While microglia are traditionally considered the phagocytes of the brain, astrocytes are also capable of phagocytosis and are the most numerous cells in the brain. In Alzheimer’s disease (AD), astrocytes can be found surrounding β-amyloid (Aβ) plaques yet they seem unable to eliminate these deposits, suggesting phagocytosis may be impaired in AD. Mechanisms that might diminish astrocyte phagocytosis in AD are currently unclear. Here, we demonstrate that the autophagy protein beclin 1, which is reduced in AD, plays a role in regulating astrocyte phagocytosis. Specifically, we show that reducing beclin 1 in C6 astrocytes impairs the phagocytosis of latex beads, reduces retromer levels, and impairs retromer recruitment to the phagosomal membrane. Furthermore, we show that these beclin 1-mediated changes are accompanied by reduced expression of the phagocytic receptor Scavenger Receptor Class B type I (SR-BI). Collectively, these findings suggest a critical role for the protein beclin 1 in both receptor trafficking and receptor-mediated phagocytosis in astrocytes. Moreover, these findings provide insight into mechanisms by which astrocytes may become impaired in AD.

Reaction of different cell types of the brain on neurotoxin cuprizone and hormone melatonin treatment in young and aging mice.

The brain myelin and neurons destruction in multiple sclerosis may be associated with the production of neuroinflammatory cells (macrophages, astrocytes, T-lymphocytes) of pro-inflammatory cytokines and free radicals. The age-associated changes of the above cells can influence on the response of nervous system cells to toxic damaging and regulatory factors of humoral/endocrine nature, in particular pineal hormone melatonin. The study aim was (1) to evaluate changes of the brain macrophages, astrocytes, T-cells, neural stem cells, neurons, and central nervous system (CNS) functioning in the neurotoxin cuprizone-treated mice of different age; and (2) to assess in such mice the effects of exogenous melatonin and possible courses of its action. A toxic demyelination and neurodegeneration model was induced in 129/Sv mice aged 3-5 and 13-15 months by adding cuprizone neurotoxin to their food for 3 weeks. From the 8th day of the cuprizone treatment, melatonin was injected intraperitoneally at 6 p.m. daily, at a dose of 1 mg/kg. The brain GFPA + -cells were evaluated by immunohistochemical method, the proportion of CD11b+, CD3+CD11b+, CD3+, CD3+CD4+, CD3+CD8+, Nestin+-cells was determined via flow cytometry. Macrophage activity was evaluated by their ability to phagocytose latex beads Morphometric analysis of the brain neurons and the behavioral reactions ("open field" and rotarod tests) were performed. To assess the involvement of the bone marrow and thymus in the action of melatonin, the amount of granulocyte/macrophage colony-forming cells (GM-CFC), and blood monocytes and thymic hormone thymulin were evaluated. The numbers of the GFAP+-, CD3+-, CD3+CD4+, CD3+CD8+, CD11b+, CD3+CD11b+, Nestin+-cells and macrophages phagocytic latex beads and malondialdehyde (MDA) content were increased in the brain of young and aging mice under cuprizone influence. The proportion of undamaged neurons within the brain, motor, affective, and exploratory activities, and muscle tone decreased in mice of both ages. Introducing melatonin to mice of any age reduced the number of GFAP+-, CD3+- cells and their subpopulations, macrophage activation, and MDA content. At the same time, the percentage of brain neurons that were unchanged increased as the number of Nestin+ cells decreased. The behavioral responses were also improved. Besides, the number of bone marrow GM-CFC and the blood level of monocytes and thymulin increased. The effects of both neurotoxin and melatonin on the brain astrocytes, macrophages T-cells, and immune system organs as well as the structure and functioning of neurons were more pronounced in the young mice. We have observed the involvement of the astrocytes, macrophages, T-cells, neural stem cells, and neurons in the brain reaction of mice different age after administration of neurotoxin cuprizone and melatonin. The brain cell composition reaction has the age features. The neuroprotective effects of melatonin in cuprizone-treated mice have been realized through an improvement of the brain cell composition and oxidative stress factors and functioning of bone marrow and thymus.

Phagocytosis of latex beads by a human monocytic Mono Mac 6 cell line and effects of low-frequency electromagnetic field interaction.

Some studies have shown that electromagnetic fields (EMFs) may impact immune response cells and their functions. The first stage of the defense from pathogens is innate immunity encompassing phagocytosis and phagocytosis-related intracellular effects. Our work aimed to determine the influence of a low-frequency electromagnetic field (7 Hz, 30 mTrms) on the phagocytosis process of latex beads (LBs), the production of reactive oxygen species (ROS), and viability changes in a human monocytic Mono Mac 6 (MM6) cell line as an experimental model of the phagocytosing cells in in vitro cell culture conditions. For these purposes, cells were firstly activated with infectious agents such as lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB), or the proliferatory agent phytohaemagglutinin (PHA), and then a phagocytosis test was performed. Cell viability and range of phagocytosis of latex beads by MM6 cells were measured by flow cytometry, and the level of ROS was evaluated with the use of a cytochrome C reduction test. The obtained results revealed that applied EMF exposure mainly increased the necrosis parameter of cell death when they were pre-stimulated with SEB as an infectious factor and subsequently phagocytosed LBs (P=0.001). Prestimulation with other agents like LPS or PHA preceding phagocytosis resulted in no statistically significant changes in cell death parameters. The level of ROS depended on the used stimulatory agent, phagocytosis, and/or EMF exposure. The obtained effects for EMF exposure indicated only a slight decrease in the ROS level for cells phagocytosing latex beads and being treated with SEB or PHA, while the opposite effect was observed for LPS pre-stimulated cells (data not statistically significant). The results concerning the viability of phagocytosing cells, the effectiveness of the phagocytosis process, and the level of radical forms might result from applied EMF parameters like signal waveform, frequency, flux density, and especially single EMF exposure.

Functional homology of tachykinin signalling: The influence of human substance P on the immune system of the mealworm beetle, Tenebrio molitor L.

Tachykinin-related peptides (TRPs) are one of the most prominent families of neuropeptides in the animal kingdom. Insect TRPs display strong structural and functional homology to vertebrate tachykinins (TKs). To study functional homologies between these two neuropeptide families, the influence of human substance P (SP, one of the essential vertebrate TKs) on the immune system of the mealworm beetle, Tenebrio molitor L., was analysed.Human SP influences the phagocytic abilities of T. molitor haemocytes. Peptide injection leads to an increase in the number of haemocytes participating in the phagocytosis of latex beads. In contrast, incubation of haemocytes from non-injected beetles in a solution of physiological saline and SP causes a decrease in phagocytic activity. Treatment with human SP also led to increased adhesion of haemocytes, but no changes in the arrangement of the F-actin cytoskeleton were observed. Interestingly, 6 h after human SP injection, increased DNA integrity in T. molitor haemocytes was reported. The opposite effects were observed 24 h after SP injection.Human SP caused the upregulation of humoral immune responses, such as phenoloxidase (PO) activity in the T. molitor haemolymph, and the downregulation of immune-related genes encoding coleoptericin A, tenecin 3 and Toll receptor. However, genes encoding attacin 2 and cecropin were upregulated. Despite these differences, the antimicrobial activity of T. molitor haemolymph was significantly lower in beetles injected with SP than in control beetles. Moreover, an analysis of the direct influence of SP on lysozyme activity was performed. Our results suggest that SP at a concentration of 10−6 M can directly inhibit lysozyme activity. However, an opposite effect was reported after the application of SP at a concentration of 10−4 M.The presented results suggest structural and functional homology between TK signalling in vertebrates and insects. Primarily, this was visible in the context of the humoral response and general antimicrobial activity of T. molitor haemolymph. However, some of the results related to haemocyte function may also indicate the importance of the TK and TRP sequences for evoking immunological effects.

Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages.

The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. To interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc. We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement fragment C3a. Although it is known that complement components react with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage interactions with fungi has not been elucidated. We demonstrated that whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc, the ubiquitous fungal pathogen Candida albicans, and the causative agent of Valley Fever Coccidioides posadasii. We showed that C3aR localizes to the early phagosome during Hc infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc-macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi.

Leishmania infection-induced multinucleated giant cell formation via upregulation of ATP6V0D2 expression.

Leishmaniasis is caused by infection with protozoan parasites of the genus Leishmania. In both clinical and experimental visceral leishmaniasis, macrophage multinucleation is observed in parasitized tissues. However, the feature and the mechanism of macrophage multinucleation remained unclear. Here, we report that infection of Leishmania donovani, a causative agent of visceral leishmaniasis, induces multinucleation of bone marrow-derived macrophages (BMDMs) in vitro. When these infection-induced multinucleated macrophages were compared with cytokine-induced multinucleated giant cells, the former had higher phagocytic activity on red blood cells but no apparent changes on phagocytosis of latex beads. BMDMs infected with L. donovani had increased expression of ATP6V0D2, one of the components of V-ATPase, which was also upregulated in the spleen of infected mice. Infection-induced ATP6V0D2 localized in a cytoplasmic compartment, which did not overlap with the mitochondria, endoplasmic reticulum, or lysosomes. When ATP6V0D2 expression was recombinantly induced in BMDMs, the formation of multinucleated macrophages was induced as seen in the infected macrophages. Taken together, L. donovani infection induces multinucleation of macrophages via ATP6V0D2 upregulation leading to a unique metamorphosis of the macrophages toward hemophagocytes.

Protein expression changes during phagocytosis influenced by low-frequency electromagnetic field exposure

The aim of our studies was to determine the influence of a low-frequency electromagnetic field (EMF) on the phagocytosis of latex beads (LBs) and the expression level of proteins/genes in the human monocytic macrophage Mono Mac 6 (MM6) cell line in in vitro conditions. Before phagocytosis assay cells were pre-stimulated with infectious agents such as lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB), or the proliferatory agent phytohaemagglutinin (PHA), and then exposed to EMF (30 mT, 7 Hz, 3 h). The expression of cytoplasmic proteins like iPLA, cPLA, iNOS, NLR3/4, and Hsp70 involved in the immune response pathways to phagocytosed particles were evaluated with the usage of the Western blot analysis. mRNA encoding the iNOS protein was detected by reverse transcription PCR method. The most meaningful changes were observed for PLA2 and NLC4 proteins level and between iNOS protein expression and mRNA encoding iNOS protein amount. The EMF exposure exerted the strongest effect on iNOS encoding mRNA in cells pre-stimulated with LPS or SEB and phagocytosing LBs. The influence of EMF on phagocytosis was experimentally proved for the first time and there is a need for further investigations in term of the usage of EMF as a prospect, supportive therapy.

Anti-inflammatory role of GM1 and other gangliosides on microglia

BackgroundGangliosides are glycosphingolipids highly enriched in the brain, with important roles in cell signaling, cell-to-cell communication, and immunomodulation. Genetic defects in the ganglioside biosynthetic pathway result in severe neurodegenerative diseases, while a partial decrease in the levels of specific gangliosides was reported in Parkinson’s disease and Huntington’s disease. In models of both diseases and other conditions, administration of GM1—one of the most abundant gangliosides in the brain—provides neuroprotection. Most studies have focused on the direct neuroprotective effects of gangliosides on neurons, but their role in other brain cells, in particular microglia, is not known. In this study we investigated the effects of exogenous ganglioside administration and modulation of endogenous ganglioside levels on the response of microglia to inflammatory stimuli, which often contributes to initiation or exacerbation of neurodegeneration.MethodsIn vitro studies were performed using BV2 cells, mouse, rat, and human primary microglia cultures. Modulation of microglial ganglioside levels was achieved by administration of exogenous gangliosides, or by treatment with GENZ-123346 and L–t-PDMP, an inhibitor and an activator of glycolipid biosynthesis, respectively. Response of microglia to inflammatory stimuli (LPS, IL-1β, phagocytosis of latex beads) was measured by analysis of gene expression and/or secretion of pro-inflammatory cytokines. The effects of GM1 administration on microglia activation were also assessed in vivo in C57Bl/6 mice, following intraperitoneal injection of LPS.ResultsGM1 decreased inflammatory microglia responses in vitro and in vivo, even when administered after microglia activation. These anti-inflammatory effects depended on the presence of the sialic acid residue in the GM1 glycan headgroup and the presence of a lipid tail. Other gangliosides shared similar anti-inflammatory effects in in vitro models, including GD3, GD1a, GD1b, and GT1b. Conversely, GM3 and GQ1b displayed pro-inflammatory activity. The anti-inflammatory effects of GM1 and other gangliosides were partially reproduced by increasing endogenous ganglioside levels with L–t-PDMP, whereas inhibition of glycolipid biosynthesis exacerbated microglial activation in response to LPS stimulation.ConclusionsOur data suggest that gangliosides are important modulators of microglia inflammatory responses and reveal that administration of GM1 and other complex gangliosides exerts anti-inflammatory effects on microglia that could be exploited therapeutically.

Immunomodulatory effect of petroleum ether extract and ethyl acetate fraction of bengkoang (Pachyrhizus erosus (L.) Urban) in vitro

Bengkoang (Pachyrhizus erosus (L.) Urban) contains phytosterol and the isoflavone daidzein, which are thought to have immunomodulatory activity. There have been no studies reporting on the immunomodulatory effects of bengkoang extract containing polar and semi-polar compounds, such as phytosterols and isoflavone-like compounds. The objective of this study was to evaluate the immunomodulatory effects of bengkoang extracts, including petroleum ether extract (PEE), methanol extract (ME), and the ethyl acetate fraction (EAF) of bengkoang, in vitro. The immunomodulatory effects of PEE, ME, and EAF of bengkoang were determined according to the phagocytic activity of macrophages based on phagocytosis of latex beads, lymphocyte proliferation, and detection of cytokine production of tumor necrosis factor-α (TNF-α) interleukin-6 (IL-6), and interleukin-10 (IL-10) levels. Results: The phagocytic index and phagocytic capacity of ME, PEE, and EAF of bengkoang on macrophage cells were significantly increased (p < 0.05), whereas lymphocyte proliferation was unchanged compared with the control (p > 0.05), and ME of bengkoang enhanced the levels of the cytokines TNF-α and IL-6. In contrast, PEE and EAF of bengkoang decreased TNF-α and IL-6 levels compared with the control group. All of the bengkoang extracts decrease the production of the anti-inflammatory cytokine IL-10. In conclusion, this study showed that PEE, ME, and EAF of bengkoang could increase the non-specific immune response (phagocytic activity) but had a lesser effect on the specific immune response (lymphocyte proliferation). The ME of bengkoang acts as an immunostimulant by increasing the levels of the inflammatory cytokines TNF-α and IL-6 and decreasing those of the anti-inflammatory cytokine IL-10.

Involvement of Rac1 in macrophage activation by brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration. Tissue regeneration is characterized by inflammation, which directs the quality of tissue repair. This study aimed to investigate the effect of BDNF on the phagocytic activity of RAW264.7 cells. In addition, we studied the effect of BDNF on guanosine triphosphatase (GTP)-RAS-related C3 botulinus toxin substrate (Rac)1 and phospho-Rac1 levels in RAW264.7 cells. Rac1 inhibitor inhibited BDNF-induced phagocytosis of latex-beads. In addition, BDNF enhanced Porphyromonas gingivalis (Pg) phagocytosis by RAW264.7 cells as well as latex-beads. We demonstrated for the first time that BDNF enhances phagocytic activity of RAW264.7 cells through Rac1 activation. The present study proposes that BDNF may reduce inflammatory stimuli during BDNF-induced periodontal tissue regeneration through enhanced phagocytic activity of macrophages.

Mechanosensing of Substrate Stiffness Regulates Effector Functions of Human Neutrophils

During a response to injury or infection, neutrophils (PMNs) navigate through a tissue microenvironment that is densely packed with parenchymal cells and extracellular matrix. Within these tissues, PMNs sense the physical property of elasticity as a regulatory element as it pertains to motility and effector functions. Historically, rigid tissue culture surfaces like glass and plastic have been used to study many key anti-microbial effector functions of PMNs; however, these fail to mimic the elasticity of peripheral tissues. We hypothesize that elasticity plays a key regulatory role for PMN anti-microbial effector functions associated with pathogen clearance such as phagocytosis, NETosis, respiratory burst, and degranulation. When comparing phagocytosis by neutrophils on soft substrates and stiff substrates coated with fibronectin, we found that stiffness is selectively regulatory. While Zymosan and Whole Glucan Particles were more readily phagocytosed on soft substrates, IgG-opsonized latex beads were more efficiently taken up on stiff substrates. Further, C3bi-opsonized latex beads and apoptotic bodies were phagocytosed equivalently on all stiffnesses. In addition to a mechanosensitive response to phagocytosed particles, we also saw differential mechanosensitive responses in their mechanism. For example, on stiff substrates, inhibiting Rho-associated protein kinase reduced phagocytosis of Zymosan, while blocking CD45 reduced phagocytosis of IgG-opsonized latex beads. Further, Zymosan uptake was found to increase on soft substrates when actin-related protein Arp2/3 complex was blocked, but have no effect on stiff substrates, further exaggerating the soft/stiff phenotype; conversely, blocking formin-mediated actin had the opposite effect and lowered phagocytosis of Zymosan on soft substrates, attenuating the soft/stiff phenotype. Finally, not all blocking targets showed mechanosensitive responses; while inhibition of Spleen Tyrosine Kinase had no effect on Zymosan uptake on any stiffness, blocking total actin polymerization reduced uptake on all stiffnesses. Taken together, these findings show a highly complex mechanosensitive response to phagocytosis in regards to both particle type and mechanism of uptake. Further, PMNs seeded on Candida albicans hyphae were mechanosensitive in regards to oxidative burst and NETosis; both ROS production and NETosis increased on stiffer substrates. Finally, in addition to phagocytosis and hyphae, we also looked at cytokine release to secondary stimulation by fMLP. We found both Lactoferrin and IL-8 release to increase on stiffer substrates, while elastase release was not mechanosensitive. Taken together, these findings indicate a strong regulatory role of substrate stiffness during PMN effector responses. While extensive work has been done to elucidate PMN biochemical interactions, understanding important mechanosensitive mechanisms underlying phagocytosis, NETosis, respiratory burst, and cytokine release may provide further therapeutic targets to benefit patients in the future by reducing harmful PMN behaviors and/or promoting behaviors crucial to pathogen clearance.

Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.