Immunomodulatory Activity of Octenyl Succinic Anhydride Modified Porang (Amorphophallus oncophyllus) Glucomannan on Mouse Macrophage-Like J774.1 Cells and Mouse Primary Peritoneal Macrophages.

Porang is a local plant of Indonesia, which has a high content of glucomannan. In this study, porang glucomannan (PG) was esterified with octenyl succinic anhydride (OSA) to enhance emulsion properties to be widely used in food industry. OSA-modified PG (OSA-PG) enhanced the phagocytosis activity of macrophage-like J774.1 cells and mouse peritoneal macrophages. In addition, OSA-PG increased the production of IL-6 and TNF-α by enhancing their gene expression. Immunoblot analysis displayed that OSA-PG tended to activate both nuclear factor-κB and mitogen-activated protein kinase cascades. Treatment of OSA-PG with polymyxin B revealed that cytokine production induced by OSA-PG was not caused by endotoxin contamination. Our findings also indicated that OSA-PG activates macrophages through not only Toll-like receptor (TLR) 4, but another receptor. Overall findings suggested that OSA-PG has a potential as an immunomodulatory food factor by stimulating macrophages.

Immunostimulatory effect of aqueous extract of Coriandrum sativum L. seed on macrophages.

BACKGROUND: Coriandrum sativum L. seed is generally used as a spice and crude drug. Although many functions of the various components in C. sativum L. seed have been reported, the immunostimulatory effect of water-soluble components in C. sativum L. seed has not been studied. In the present study, we focused on the immunostimulatory effect of C. sativum L. seed aqueous extract (CAE) on macrophages as a novel health function of C. sativum L. seed components. RESULTS: CAE significantly enhanced the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in both RAW264.7 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. CAE also stimulated nitric oxide (NO) production and the phagocytosis activity in RAW264.7 cells. We suggest that the activity of CAE is a result of the upregulation of mitogen-activated protein kinase and nuclear factor-κB cascades via TLR4. In addition, IL-6 production by peritoneal macrophages collected from CAE-administered mice was significantly enhanced, suggesting that CAE could stimulate macrophage activity in vivo. CONCLUSION: The findings of the present study suggest that CAE contains a novel water-soluble component with an immunostimulatory effect on macrophages. CAE would contribute to activating host defense against pathogens by stimulating the innate immunity. © 2017 Society of Chemical Industry.

Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages.

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.

In vitro immunotoxicology of quantum dots and comparison with dissolved cadmium and tellurium.

The increasing use of products derived from nanotechnology has raised concerns about their potential toxicity, especially at the immunocompetence level in organisms. This study compared the immunotoxicity of cadmium sulfate/cadmium telluride (CdS/Cd-Te) mixture quantum dots (QDs) and their dissolved components, cadmium chloride (CdCl2 )/sodium telluride (NaTeO3 ) salts, and a CdCl2 /NaTeO3 mixture on four animal models commonly used in risk assessment studies: one bivalve (Mytilus edulis), one fish (Oncorhynchus mykiss), and two mammals (mice and humans). Our results of viability and phagocytosis biomarkers revealed that QDs were more toxic than dissolved metals for blue mussels. For other species, dissolved metals (Cd, Te, and Cd-Te mixture) were more toxic than the nanoparticles (NPs). The most sensitive species toward QDs, according to innate immune cells, was humans (inhibitory concentration [IC50 ] = 217 µg/mL). However, for adaptative immunity, lymphoblastic transformation in mice was decreased for small QD concentrations (EC50 = 4 µg/mL), and was more sensitive than other model species tested. Discriminant function analysis revealed that blue mussel hemocytes were able to discriminate the toxicity of QDs, Cd, Te, and Cd-Te mixture (Partial Wilk's λ = 0.021 and p < 0.0001). For rainbow trout and human cells, the immunotoxic effects of QDs were similar to those obtained with the dissolved fraction of Cd and Te mixture. For mice, the toxicity of QDs markedly differed from those observed with Cd, Te, and dissolved Cd-Te mixture. The results also suggest that aquatic species responded more differently than vertebrates to these compounds. The results lead to the recommendation that mussels and mice were most able to discriminate the effects of Cd-based NPs from the effects of dissolved Cd and Te at the immunocompetence level.

SpBark Suppresses Bacterial Infection by Mediating Hemocyte Phagocytosis in an Invertebrate Model, Scylla paramamosain.

Scavenger receptors are cell surface membrane-bound receptors that typically bind multiple ligands and promote the removal of endogenous proteins and pathogens. In this study, we characterized a novel scavenger receptor-like protein, namely, SpBark. SpBark was upregulated in hemocytes after challenges with bacteria, suggesting that it might be involved in antibacterial defense. SpBark is a type I transmembrane protein with four extracellular domains, including three scavenger receptor cysteine-rich domains (SRCRDs) and a C-type lectin domain (CTLD). Western blot assay showed that SpBark CTLD possessed a much stronger binding activity to tested microbes than the three SRCRDs. It also exhibited apparent binding activities to lipopolysaccharide (LPS) and acetylated low-density lipoprotein (ac-LDL), whereas the other SRCRDs showed much lower or no binding activities to these components. Agglutination activities were observed in the presence of Ca2+ by incubating microorganisms with SpBark CTLD instead of SRCRDs. These results suggested that SpBark CTLD was the major binding site for ac-LDL and LPS. Coating Vibrio parahemolyticus with SpBark CTLD promoted bacterial clearance in vivo. This finding indicated that SpBark might participate in the immune defenses against Gram-negative bacteria through a certain mechanism. The promotion of bacterial clearance by SpBark was further determined using SpBark-silenced crabs injected with V. parahemolyticus. SpBark knockdown by injection of SpBark dsRNA remarkably suppressed the clearance of bacteria in hemolymph. Meanwhile, it also severely restrained the phagocytosis of bacteria. This finding suggested that SpBark could modulate the phagocytosis of bacteria, and the promotion of bacterial clearance by SpBark was closely related to SpBark-mediated phagocytosis activity. The likely mechanism of bacterial clearance mediated by SpBark was as follows: SpBark acted as a pattern recognition receptor, which could sense and bind to LPS on the surface of invading bacteria with its CTLD in hemolymph. The binding to LPS made the bacteria adhere to the surface of hemocytes. This process would facilitate phagocytosis of the bacteria, resulting in their removal. This study provided new insights into the hemocyte phagocytosis mechanisms of invertebrates and the multiple biological functions of Bark proteins.

Immunostimulatory effect of dried bonito extract on mouse macrophage cell lines and mouse primary peritoneal macrophages.

Dried bonito is a preserved food used in Japan, which contains abundant flavor ingredients and functional substances. We focused on the immunostimulatory effect of dried bonito extract (DBE) on mouse macrophage-like J774.1 cells, RAW264.7 cells, and mouse primary peritoneal macrophages. DBE significantly stimulated the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by both J774.1 cells and peritoneal macrophages by enhancing the cytokine gene expression levels. In addition, DBE stimulated nitric oxide production by enhancing the expression of inducible nitric oxide synthase in RAW264.7 cells. DBE also increased the phagocytosis activity of J774.1 cells. Immunoblot analysis revealed that DBE has an immunostimulatory effect on macrophages through activation of mitogen-activated protein kinase and nuclear factor-κB cascades. TNF-α production enhanced by DBE was partially inhibited by treatment with TLR4 inhibitor TAK-242, whereas IL-6 production enhanced by DBE was almost inhibited. These results suggested that DBE is thought to strongly stimulate the TLR4 signaling pathway for macrophage activation, and its activation is also involved in other signaling. Finally, the phagocytosis activity of peritoneal macrophages from DBE-administered BALB/c mice increased significantly, suggesting that DBE has the potential to stimulate macrophage activity in vivo. In conclusion, these data indicated that DBE contributes to activating host defense against pathogens by activating innate immunity.

The primary culture of carp (Cyprinus carpio) macrophages and the verification of its phagocytosis activity.

This study establishes the primary culture method for red carp (Cyprinus carpio) macrophages in vitro and lays the foundation for further research in the fish immune system. The healthy adult red carp was chosen, and mechanical separation and cell adherent culture methods were used to isolate the primary macrophages. Compared to the traditional method of Percoll discontinuous density gradient isolation, the protocol we reported here makes cell isolation steps more concise and obtains more healthy cells with high macrophage purity. The cells were uniform in size with a clearly visible nucleus. Trypan blue staining and non-radioactive cell proliferation assay were used to detect the cell survival rate. Further, we provide optimum culture conditions which include cell density (1 × 10(7) cells/mL), culture medium (Leibovitz's L-15), pH (7.2-7.4), temperature (26°C), and adherent time (24 h). Macrophages have been identified by nonspecific esterase and Wright-Giemsa staining and have shown to grow very well. In addition, the macrophages have a very strong bactericidal activity against three kinds of bacteria, further verifying good growth conditions and proper function.

Detection of immunotoxic effects of estrogenic and androgenic endocrine disrupting compounds using splenic immune cells of the female three-spined stickleback, Gasterosteus aculeatus (L.).

Today, the list of endocrine disrupting compounds (EDCs) in freshwater and marine environments that mimic or block endogenous hormones is expanding at an alarming rate. As immune and reproductive systems may interact in a bidirectional way, some authors proposed the immune capacities as attractive markers to evaluate the hormonal potential of environmental samples. Thus, the present work proposed to gain more knowledge on direct biological effects of natural and EDCs on female fish splenic leucocyte non-specific immune activities by using ex vivo assays. After determining the optimal required conditions to analyze splenic immune responses, seven different EDCs were tested ex vivo at 0.01, 1 and 100nM over 12h on the leucocyte functions of female three-spined stickleback, Gasterosteus aculeatus. In summary, we found that natural hormones acted as immunostimulants, whilst EDCs were immunosuppressive.

Immunomodulatory effects of selected Malaysian plants on the CD18/11a expression and phagocytosis activities of leukocytes

Objective: To investigate the effects of 20 methanolic extracts from Malaysian selected plants on CD18/11a expression and phagocytosis activity of leukocytes using flow cytometry analysis. Methods: The effects of methanolic extracts on CD18/11a expression and phagocytosis of leukocytes were measured by labelling the cells with CD18-fluorescein isothiocyanate and ingestion labelled with Escherichia coli-fluorescein isothiocyanate and then analyzed using flow cytometer. Results: About 12 out of 20 methanolic extracts of selected Malaysian medicinal plants significantly (P≤0.05) inhibited the CD18/11a expression of leukocytes at both concentrations of 6.25 μg/mL and 100 μg/mL in dose dependent manner. The most active inhibitory was shown in Citrus aurantifolia (Christm.) Swingle and Alpinia galangal (L.) Willd. at dosage 100 μg/mL. Moreover, the Orthosiphon aristatus (Blume) Miq (O. aristatus). showed the highest stimulatory activity at the concentration of 100 μg/mL. Other than that, four plant extracts significantly (P≤0.05) rose the phagocytosis activities of leukocytes in dose dependent manner. However, Annona muricata L. and O. aristatus showed the highest stimulated activities at the 100 μg/mL concentration. Conclusions: The results suggest that methanolic extracts of Citrus aurantifolia, Alpinia galangal, O. aristatus and Annona muricata are able to modulate innate immune system and can potentially be recognized as therapeutic agents for modulating immune system.