Abstract:Alginate from marine seaweeds is receiving continuous attention owing to its wide physiological activities. Herein, we sought to elucidate possible effects of alginate-derived polyguluronate (PG) and unsaturated guluronate oligosaccharide (GOS) on antibacterial activities of macrophages. Our results showed that, in contrast to PG, GOS markedly increased the phagocytosis of IgG-opsonized Escherichia coli and Staphylococcus aureus and further inhibited the survival of intracellular bacteria in macrophages. In li… Show more
“…2C). Considering that GOS cannot alter CD14 expression in macrophages, which has been demonstrated by our previous publication 20 , we speculate that GOS could activate TLR4 and MD2, and trigger the MyD88-dependent pathway, leading to the production of pro-inflammatory cytokines without a requirement for CD14.Figure 2GOS increases TLR4 expression and stimulates Akt/mTOR activation in RAW264.7 cells. ( A ) RAW264.7 cells were treated with 1 mg/ml GOS for 24 h, and TLR4 expression was evaluated by immunofluorescence analysis using an anti-TLR4 antibody.…”
Section: Resultsmentioning
confidence: 61%
“…GOS has been found to augment the release of cytokines, such as tumour necrosis factor-α (TNF-α) 16 , and to induce the production of nitric oxide (NO) by activating NF-κB and MAPK signalling pathways 19 in RAW264.7 cells. GOS also promotes macrophage bactericidal activity by increasing phagocytosis in vitro and in vivo
20 . Furthermore, proteomic analysis revealed that GOS improves macrophage homeostasis 21 .…”
Alginate, a natural acidic polysaccharide extracted from marine brown seaweeds, is composed of different blocks of β-(1, 4)-D-mannuronate (M) and its C-5 epimer α-(1, 4)-L-guluronate (G). Alginate-derived guluronate oligosaccharide (GOS) readily activates macrophages. However, to understand its role in immune responses, further studies are needed to characterize GOS transport and signalling. Our results show that GOS is recognized by and upregulates Toll-like receptor 4 (TLR4) on RAW264.7 macrophages, followed by its endocytosis via TLR4. Increased expression of TLR4 and myeloid differentiation protein 2 (MD2) results in Akt phosphorylation and subsequent activation of both nuclear factor-κB (NF-κB) and mechanistic target of rapamycin (mTOR). Moreover, GOS stimulates mitogen-activated protein kinases (MAPKs); notably, c-Jun N-terminal kinase (JNK) phosphorylation depends on TLR4 initiation. All these events contribute to the production of inflammatory mediators, either together or separately. Our findings also reveal that GOS induces cytoskeleton remodelling in RAW264.7 cells and promotes macrophage proliferation in mice ascites, both of which improve innate immunity. Conclusively, our investigation demonstrates that GOS, which is dependent on TLR4, is taken up by macrophages and stimulates TLR4/Akt/NF-κB, TLR4/Akt/mTOR and MAPK signalling pathways and exerts impressive immuno-stimulatory activity.
“…2C). Considering that GOS cannot alter CD14 expression in macrophages, which has been demonstrated by our previous publication 20 , we speculate that GOS could activate TLR4 and MD2, and trigger the MyD88-dependent pathway, leading to the production of pro-inflammatory cytokines without a requirement for CD14.Figure 2GOS increases TLR4 expression and stimulates Akt/mTOR activation in RAW264.7 cells. ( A ) RAW264.7 cells were treated with 1 mg/ml GOS for 24 h, and TLR4 expression was evaluated by immunofluorescence analysis using an anti-TLR4 antibody.…”
Section: Resultsmentioning
confidence: 61%
“…GOS has been found to augment the release of cytokines, such as tumour necrosis factor-α (TNF-α) 16 , and to induce the production of nitric oxide (NO) by activating NF-κB and MAPK signalling pathways 19 in RAW264.7 cells. GOS also promotes macrophage bactericidal activity by increasing phagocytosis in vitro and in vivo
20 . Furthermore, proteomic analysis revealed that GOS improves macrophage homeostasis 21 .…”
Alginate, a natural acidic polysaccharide extracted from marine brown seaweeds, is composed of different blocks of β-(1, 4)-D-mannuronate (M) and its C-5 epimer α-(1, 4)-L-guluronate (G). Alginate-derived guluronate oligosaccharide (GOS) readily activates macrophages. However, to understand its role in immune responses, further studies are needed to characterize GOS transport and signalling. Our results show that GOS is recognized by and upregulates Toll-like receptor 4 (TLR4) on RAW264.7 macrophages, followed by its endocytosis via TLR4. Increased expression of TLR4 and myeloid differentiation protein 2 (MD2) results in Akt phosphorylation and subsequent activation of both nuclear factor-κB (NF-κB) and mechanistic target of rapamycin (mTOR). Moreover, GOS stimulates mitogen-activated protein kinases (MAPKs); notably, c-Jun N-terminal kinase (JNK) phosphorylation depends on TLR4 initiation. All these events contribute to the production of inflammatory mediators, either together or separately. Our findings also reveal that GOS induces cytoskeleton remodelling in RAW264.7 cells and promotes macrophage proliferation in mice ascites, both of which improve innate immunity. Conclusively, our investigation demonstrates that GOS, which is dependent on TLR4, is taken up by macrophages and stimulates TLR4/Akt/NF-κB, TLR4/Akt/mTOR and MAPK signalling pathways and exerts impressive immuno-stimulatory activity.
“…Cells were incubated with Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) at 37 °C and 5% CO 2 in a humidified environment. For bacterial inoculation into macrophage cells we followed similar protocols whereby phagocytosis was observed14. Colonies of EGFP expressing E. coli were transferred from the LB agar plate to a 14 mL round bottom tube filled with 3 mL of LB broth and 50 μ g/mL ampicillin medium for 20 hours at 37 °C and 225 rpm.…”
Section: Methodsmentioning
confidence: 99%
“…Other examples include expression of fluorescent proteins fused to the LC-3 protein during the late phagosome-lysosome-autophagosome merging processes2. It is also common to fluorescently label the target pathogen; Xu and colleagues studied phagocytic activity by expressing fluorescent proteins in bacteria which were subsequently introduced into mammalian cultures14. Similarly, Oh et al .…”
mentioning
confidence: 99%
“…Moreover, bacterial species might escape the phagosome owing to their ability to withstand the acidic microenvironment, as shown with Förster Resonance Energy Transfer (FRET) experiments23. Thus phagocytic-based research is increasingly dependent on multiple fluorescence labels, full spectral measurements, complex data analysis, efficient sample-screening, fluorescence ratio-metric methods and combinations of spectrofluorometry, fluorescence microscopy and flow cytometry9131415172425.…”
Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.
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