Phytocannabinoids Reduce Inflammation of Primed Macrophages and Enteric Glial Cells: An In Vitro Study.

Intestinal inflammation is mediated by a subset of cells populating the intestine, such as enteric glial cells (EGC) and macrophages. Different studies indicate that phytocannabinoids could play a possible role in the treatment of inflammatory bowel disease (IBD) by relieving the symptoms involved in the disease. Phytocannabinoids act through the endocannabinoid system, which is distributed throughout the mammalian body in the cells of the immune system and in the intestinal cells. Our in vitro study analyzed the putative anti-inflammatory effect of nine selected pure cannabinoids in J774A1 macrophage cells and EGCs triggered to undergo inflammation with lipopolysaccharide (LPS). The anti-inflammatory effect of several phytocannabinoids was measured by their ability to reduce TNFα transcription and translation in J774A1 macrophages and to diminish S100B and GFAP secretion and transcription in EGCs. Our results demonstrate that THC at the lower concentrations tested exerted the most effective anti-inflammatory effect in both J774A1 macrophages and EGCs compared to the other phytocannabinoids tested herein. We then performed RNA-seq analysis of EGCs exposed to LPS in the presence or absence of THC or THC-COOH. Transcriptomic analysis of these EGCs revealed 23 differentially expressed genes (DEG) compared to the treatment with only LPS. Pretreatment with THC resulted in 26 DEG, and pretreatment with THC-COOH resulted in 25 DEG. To evaluate which biological pathways were affected by the different phytocannabinoid treatments, we used the Ingenuity platform. We show that THC treatment affects the mTOR and RAR signaling pathway, while THC-COOH mainly affects the IL6 signaling pathway.

Regulation of CXCR4 Expression by Taurine in Macrophage-Like Cells.

Taurine (2-aminoethanesulfonic acid) is a free ß-amino acid found at high concentrations in many mammalian tissues. Taurine plays a role in several essential biological processes, including anti-oxidation, anti-inflammation, and osmoregulation. However, its regulatory mechanisms, especially at the genetic and molecular levels, have not been elucidated. Here, we targeted immune-related genes and investigated the effects of taurine on immune-related gene expression in macrophage-like cells. J774.1 cell line was used, and the effect of taurine on mRNA expression of immune-related genes such as cytokines, their receptors, and toll-like receptors was examined. Among these, taurine significantly increased the mRNA levels of C-X-C chemokine receptor 2 (CXCR2), chemokine receptor. Furthermore, we found that the taurine-induced increase in CXCR4 mRNA levels was higher than that in CXCR2 mRNA levels. Taurine increased both mRNA and protein expression levels of CXCR4. Additionally, we examined the effects of taurine analogs, including hypotaurine, ß-alanine, and γ-aminobutyric acid (GABA). While GABA increased the mRNA expression of CXCR4, hypotaurine slightly increased this expression, and ß-alanine had no effect, although these taurine analogs are the substrates of taurine transporter. These findings demonstrate that taurine specifically affects CXCR4 mRNA expression in macrophage-like cells.

Cytotoxicity properties of plant-mediated synthesized K-doped ZnO nanostructures.

In this study, potassium-doped zinc oxide nanoparticles (K-doped ZnO NPs) were green-synthesized using pine pollen extracts based on bioethics principles. The synthesized NPs were analyzed using X-ray diffraction (XRD), inductively coupled plasma atomic emission spectroscopy (ICP-AES), scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDXA), and transmission electron microscopy (TEM). The cytotoxicity of these nanoparticles (NPs) on normal macrophage cells and cancer cell lines was evaluated. In the same concentrations of K-doped ZnO and pure ZnO NPs, K-doped ZnO NPs demonstrated higher toxicity. The results confirmed that the doped potassium could increase cytotoxicity. The IC50 of K-doped ZnO NPs, pure ZnO NPs, and the examined control drug were 497 ± 15, 769 ± 12, and 606 ± 19 µg/mL, respectively. Considering the obtained IC50 of K-doped ZnO NPs, they were more toxic to the cancer cell lines and had less cytotoxicity on normal macrophage cells.

Anti-Inflammatory Activity of 3, 5-Diprenyl-4-hydroxyacetophenone Isolated from Ageratina pazcuarensis.

Inflammation is implicated in a wide variety of physiological and pathological processes. Plants are an important source of active anti-inflammatory compounds. The compound 3, 5-diprenyl-4-hydroxyacetophenone (DHAP) was isolated from the dichloromethane extract of the aerial parts of Ageratina pazcuarensis by chromatography and identified by spectroscopic (IR, NMR) and spectrometric (GC-MS) methods. Anti-inflammatory activity was evaluated on ear edema mouse induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) at 2 mg/ear. The antioxidant activity of DHAP was determined using DPPH assay. Cell viability was tested in J774A.1 macrophages, the levels of NO, TNF-α, IL-1ß, IL-6, and IL-10 production in macrophages stimulated with lipopolysaccharide (LPS), and membrane lysis induced by hypotonic solution in erythrocytes were evaluated. DHAP diminished the ear edema mouse in 70.10%, and it had scavenger effect against the radical with IC50 of 26.00 ± 0.37 µg/mL. Likewise, 91.78 µM of this compound inhibited the production of NO (38.96%), IL-1ß (55.56%), IL-6 (51.62%), and TNF-α (59.14%) in macrophages and increased the levels of IL-10 (61.20%). Finally, 25 and 50 µg/mL DHAP provided the greatest protection against erythrocyte membrane lysis. These results demonstrate that DHAP has anti-inflammatory activity.

Biochemical, Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin-Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios.

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin-ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.

GroEL protein of the Leptospira spp. interacts with host proteins and induces cytokines secretion on macrophages.

BACKGROUND: Leptospirosis is a zoonotic disease caused by infection with spirochetes from Leptospira genus. It has been classified into at least 17 pathogenic species, with more than 250 serologic variants. This wide distribution may be a result of leptospiral ability to colonize the renal tubules of mammalian hosts, including humans, wildlife, and many domesticated animals. Previous studies showed that the expression of proteins belonging to the microbial heat shock protein (HSP) family is upregulated during infection and also during various stress stimuli. Several proteins of this family are known to have important roles in the infectious processes in other bacteria, but the role of HSPs in Leptospira spp. is poorly understood. In this study, we have evaluated the capacity of the protein GroEL, a member of HSP family, of interacting with host proteins and of stimulating the production of cytokines by macrophages. RESULTS: The binding experiments demonstrated that the recombinant GroEL protein showed interaction with several host components in a dose-dependent manner. It was also observed that GroEL is a surface protein, and it is secreted extracellularly. Moreover, two cytokines (tumor necrosis factor-α and interleukin-6) were produced when macrophages cells were stimulated with this protein. CONCLUSIONS: Our findings showed that GroEL protein may contribute to the adhesion of leptospires to host tissues and stimulate the production of proinflammatory cytokines during infection. These features might indicate an important role of GroEL in the pathogen-host interaction in the leptospirosis.

Role of ASM/Cer/TXNIP signaling module in the NLRP3 inflammasome activation.

BACKGROUND: This study aimed to explore the effects of ceramide (Cer) on NLRP3 inflammasome activation and their underlying mechanisms. METHODS: Lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-induced NLRP3 inflammasome activation in J774A.1 cells and THP-1 macrophages was used as an in vitro model of inflammation. Western blotting and real-time PCR (RT-PCR) were used to detect the protein and mRNA levels, respectively. IL-1ß and IL-18 levels were measured by ELISA. ASM assay kit and immunofluorescence were used to detect ASM activity and Cer content. RESULTS: Imipramine, a well-known inhibitor of ASM, significantly inhibited LPS/ATP-induced activity of ASM and the consequent accumulation of Cer. Additionally, imipramine suppressed the LPS/ATP-induced expression of thioredoxin interacting protein (TXNIP), NLRP3, caspase-1, IL-1ß, and IL-18 at the protein and mRNA level. Interestingly verapamil, a TXNIP inhibitor, suppressed LPS/ATP-induced activation of TXNIP/NLRP3 inflammasome but did not affect LPS/ATP-induced ASM activation and Cer formation. TXNIP siRNA and verapamil inhibited C2-Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome. In addition, the pretreatment of cells with sulfo-N-succinimidyl oleate (SSO), an irreversible inhibitor of the scavenger receptor CD36, blocked Cer-induced upregulation of nuclear factor-κB (NF-κB) activity, TXNIP expression, and NLRP3 inflammasome activation. Inhibition of NF-κB activation by SN50 prevented Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome but did not affect CD36 expression. CONCLUSION: This study demonstrated that the ASM/Cer/TXNIP signaling pathway is involved in NLRP3 inflammasome activation. The results documented that the CD36-dependent NF-κB-TXNIP signaling pathway plays an essential role in the Cer-induced activation of NLRP3 inflammasomes in macrophages.

Extracellular Vesicles From Sporothrix brasiliensis Yeast Cells Increases Fungicidal Activity in Macrophages.

Sporotrichosis is a subcutaneous mycosis and is distributed throughout the world, although most cases belong to endemic regions with a warmer climate such as tropical and subtropical areas. The infection occurs mainly by traumatic inoculation of propagules. Similarly, to other organisms, Sporothrix brasiliensis display many biological features that aid in its ability to infect the host, such as extracellular vesicles, bilayered biological structures that provides communication between host cells and between fungi cells themselves. Recently, research on Sporothrix complex have been focused on finding new molecules and components with potential for therapeutic approaches. Here, we study the relationship among EVs and the host's macrophages as well as their role during infection to assess whether these vesicles are helping the fungi or inducing a protective effect on mice during the infection. We found that after cocultivation with different concentrations of purified yeasts EVs from Sb, J774 macrophages displayed an increased fungicidal activity (Phagocytic Index) resulting in lower colony-forming units the more EVs were added, without jeopardizing the viability of the macrophages. Interleukins IL-6, IL-10, and IL-12 were measured during the infection period, showing elevated levels of IL-12 and IL-6 in a dose-dependent manner, but no significant change for IL-10. We also assessed the expression of important molecules in the immune response, such as MHC class II and the immunoglobulin CD86. Both these molecules were overexpressed in Sb yeasts infected mice. Our results indicate that EVs play a protective role during Sporothrix brasiliensis infections.

Nanostructured lipid carrier-mediated lung targeted drug delivery system to enhance the safety and bioavailability of clofazimine.

Tuberculosis (TB) disease is caused due to the infection of Mycobacterium tuberculosis bacilli which reside in alveolar macrophages (AMs). Clofazimine (CLF) has been reinstated clinically for the treatment of TB. However, major challenge of using CLF is its severe side-effects after oral administration. The present research was aimed to establish the safety and enhance the bioavailability of CLF by loading it into nanostructured lipid carriers (CLF-NLCs) and mannosylated NLCs (M-CLF-NLCs) to selectively target the drug toward AMs. The safety of CLF-NLCs and M-CLF-NLCs was evaluated by in vitro hemocompatibility studies, cell viability studies on macrophage J774 cell lines, and in vivo acute inhalation toxicity studies. The bioavailability was estimated by single-dose pharmacokinetics and biodistribution studies. Hemocompatibility studies showed normal RBCs count and least hemolysis of 0.23 ± 0.081% for M-CLF-NLCs treated group. Cell viability studies revealed greater safety of NLCs than CLF-drug dispersion in the concentration range of 2.5-25 µg/ml. In vivo acute toxicity studies revealed no physiological or behavioral changes and no mortality recorded over 14 days period. In pharmacokinetic studies, a maximum concentration of the drug (Cmax) of 35.44 ± 0.34 µg/g from M-CLF-NLCs after 48 h and longer residence time in lung tissues observed due to its sustained release and mannose receptor-mediated endocytosis. M-CLF-NLCs showed a maximum AUC0-∞ value of 2691.83 h µg/ml in lungs that indicated twofold greater bioavailability as compared to CLF-drug dispersion. Thus, mannosylated NLCs can be used as promising carriers for the safe and effective delivery of CLF via inhalation route for the management of TB disease.

Preparation and characterization of itraconazole loaded nanomicelles based on dextran-behenic acid for cutaneous leishmaniasis treatment.

Cutaneous leishmaniasis is known as the most prevalent clinical form of leishmaniasis. It needs the development of new therapies due to the serious side-effects promoted by taking the current drugs. In the present study, dextran-behenic acid (DEX-BA) based nanomicelles were developed to direct the delivery of itraconazole (ITZ) to the macrophages and enhance its toxic effects against Leishmania parasites. DEX-BA was synthesized through the esterification of dextran with behenic acid. The critical micelle concentration of the newly developed conjugate was evaluated using pyrene as the fluorescent probe. The nanomicelles were generated by the dialysis method; then they were optimized by applying a Box-Behnken design. The effects of the dialysis temperature, polymer content, and sonication time on the characteristics of micelles were subsequently studied. Furthermore, in vitro efficacy against Leishmania major promastigotes and parasite-infected macrophages was evaluated. The optimized formulation showed the particle size of 195.16 ± 3.06 nm, the polydispersity index of 0.39 ± 0.01, the zeta potential of -16.29 ± 0.89 mV, the encapsulation efficiency % of 56.11 ± 4.9, and the release efficiency % of 51.29 ± 1.97. According to scanning electron microscopy, the nanomicelles were found to be nearly spherical in shape. ITZ-loaded nanomicelles showed the strongest anti-leishmanial activities when compared with the free ITZ and drug-free nanomicelles. It could be, therefore, concluded that ITZ-loaded nanomicelles might be useful as an alternative therapy for the treatment of cutaneous leishmania.

Cellular binding analysis of recombinant hybrid heteropolymer of camel hepcidin and human ferritin H chain. The unexpected human H-ferritin binding to J774 murine macrophage cells.

Ferritin is a molecule with enormous potentiality in biotechnology that have been already used to encapsulate molecules, as contrast in magnetic resonance imaging and to carry epitopes. We proposed to use it to carry another key protein of iron metabolism, hepcidin that is a small hormone peptide that control systemic iron homeostasis. In this work, we purified the previously produced camel hepcidin and human H-ferritin heteropolymer (HepcH-FTH) and to monitor its binding capability toward J744 cell line in presence or absence of ferric ammonium citrate. Fused camel hepcidin and human H-ferritin monomer (HepcH) as well as the assembled HepcH-FTH heteropolymer (ratio 1:5) was easily purified by a one-step purification using size exclusion chromatography. SDS-PAGE electrophoresis of HepcH, purified from soluble and insoluble fractions, showed a single band of 24 kDa with an estimated purity of at least 90%. The purification yields of HepcH from the soluble and insoluble fractions was, respectively, of about 6.80 and 2 mg/L of bacterial culture. Time curse cellular binding assays of HepcH-FTH revealed its great potential to bind the J774 cells after 15 min of incubation. Furthermore, HepcH-FTH was able to degrade ferroportin, the unique hepcidin receptor, even after 30 min of incubation with J774 cells treated with 100 µM ferric ammonium citrate. In conclusion, we proposed ferritin as a peptide carrier to promote the association of the hybrid HepcH-FTH nanoparticle with a particular type of cell for therapeutic or diagnostic.

New Hydroquinone Monoterpenoid and Cembranoid-Related Metabolites from the Soft Coral Sarcophyton tenuispiculatum.

Chemical investigation of the marine soft coral Sarcophyton tenuispiculatum resulted in the isolation of a 1,4-dihydrobenzoquinone, sarcotenuhydroquinone (1), three new cembranoids, sarcotenusenes A‒C (2‒4), and ten previously reported metabolites 5-14. The chemical structures of all isolated metabolites were determined by detailed spectroscopic analyses. In biological assays, anti-inflammatory, cytotoxic, and peroxisome proliferator-activated receptor γ (PPAR-γ) transcription factor assays of all compounds were performed. None of the isolated compounds were found to exhibit activity in the PPAR-γ transcription factor assay. The anti-inflammatory assays showed that (+)-7α,8ß-dihydroxydeepoxysarcophine (13) inhibited the production of IL-1ß to 56 ± 1% at a concentration of 30 µM in lipopolysaccharide (LPS)-stimulated J774A.1 macrophage cells. In addition, 1 and 2 were found to exhibit cytotoxicity towards a panel of cancer cell lines.

Techniques to study phagocytosis and uptake of Leishmania tarentolae by J774 macrophages.

Leishmaniasis is a vector borne parasitic disease affecting millions of people worldwide and is spreading into further areas because of global warming. The development of new active substances against these single-cell eukaryotic parasites is of great importance. Leishmania tarentolae promastigotes (LtP) are non-pathogenic for mammals and serve as model organisms for pathogenic Leishmania in basic research. However, it is important to refine methods to study the process of the infection of mammalian macrophages by LtP and pathogenic Leishmania. Important stages of the infection are phagocytosis by macrophages and multiplication of Leishmania amastigotes in the phagolysosome of macrophages. In this study, advanced methods using electron spin resonance (ESR) spectroscopy and genetically manipulated LtP were used to monitor the infection of adherent J774 macrophages with LtP. An ESR method was established to detect the formation of superoxide radicals directly in adherent J774 cells and to investigate the effect of LtP on this activity. J774 cells responded with a burst of superoxide radicals in the presence of phorbol myristate acetate as positive control. In contrast, challenging J774 cells with LtP resulted in a much lower burst of superoxide radicals. To facilitate LtP detection in the phagolysosome of J774 macrophages, LtP expressing enhanced green fluorescent protein (EGFP-LtP) were constructed. After different infection times with EGFP-LtP, the J774 cells were visualized by phase contrast microscopy and the cell number was determined. The intramacrophage Leishmania tarentolae amastigotes (LtA) expressing EGFP were detected by fluorescence microscopy and then counted with ImageJ. These experiments showed that LtP are taken up by J774 cells and form intraphagolysosomal amastigotes. LtA under our conditions multiplied intracellularly and were able to persist about 48 h in J774 cells. These experiments showed that ESR spectroscopy of attached macrophages and the use of the EGFP-LtP are suitable methods to study the initial phase of Leishmania infection in vitro.

Comparative toxicity studies on bromochloroacetate, dibromoacetate, and bromodichloroacetate in J774A.1 macrophages: Roles of superoxide anion and protein carbonyl compounds.

The brominated and mixed bromo-chloro-haloacetates, such as dibromoacetate (DBA), bromochloroacetate (BCA), and bromodichloroacetate (BDCA), are by-products of water chlorination and are found at lower levels than the fully chlorinated acetates in the drinking water. The toxicities of the compounds were assessed in J774A.1 cells and were found to induce concentration-dependent increases in cell death and superoxide anion and protein carbonyl compounds production. Compared to the previously tested concentrations of dichoroacetate (DCA) and trichloroacetate (TCA) in the same cell line, the tested haloacetates induced similar effects on cellular viability and superoxide anion production but at DBA and BCA concentrations that were approximately 40-160 times lower than those of DCA and TCA, and at BDCA concentrations that were 4-16 times lower than those of DCA and TCA. Also, production of super oxide anion, protein carbonyl compounds, and induction of phagocytic activation are suggested to play a role in their toxicity.

Eicosapentaenoic acid membrane incorporation impairs ABCA1-dependent cholesterol efflux via a protein kinase A signaling pathway in primary human macrophages.

A diet rich in n-3/n-6 polyunsaturated fatty acids (PUFAs) is cardioprotective. Dietary PUFAs affect the cellular phospholipids composition, which may influence the function of membrane proteins. We investigated the impact of the membrane incorporation of several PUFAs on ABCA1-mediated cholesterol efflux, a key antiatherogenic pathway. Arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (-28% for 70µM of EPA in HMDM), without any alterations in ABCA1 expression. These results show that PUFA membrane incorporation does not have the same consequences on cholesterol efflux from mouse and human macrophages. The EPA-treated HMDM exhibited strong phospholipid composition changes, with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which is associated with a decreased level of AA. In HMDM, EPA reduced the ATPase activity of the membrane transporter. Moreover, the activation of adenylate cyclase by forskolin and the inhibition of cAMP phosphodiesterase by isobutylmethylxanthine restored ABCA1 cholesterol efflux in EPA-treated human macrophages. In conclusion, EPA membrane incorporation reduces ABCA1 functionality in mouse macrophages as well as in primary human macrophages and this effect seems to be PKA-dependent in human macrophages.

Changes in lipid membrane mechanics induced by di- and tri-phenyltins.

Organotin compounds, being biologically active, affect a variety of cellular functions due to their ability to accumulate in and penetrate biological membranes. These compounds influence the distribution of electrostatic charges, alter organization, disrupt molecular dynamics and change mechanical properties of biological membranes. It was found that the membrane/water partition coefficient equals 4, a value significantly higher than octanol/water partition coefficient. In addition, the effect of di- and tri-phenyltin chlorides on the mechanics of model lipid membranes was measured for the first time. It has been determined that phenyltins affect the global model lipid bilayer properties by reducing the membrane expansion modulus, when measured using micromanipulation technique, and elevating the bending rigidity coefficient of the lipid bilayer, as determined with the flickering noise spectroscopy. In addition, the elevated water permeability shows that phenyltins also cause the local defects formation in the lipid bilayer, i.e. lipid pores. These data shows that phenyltins may interfere indirectly with variety cellular processes by altering non-specifically the entire cellular membrane system. Accordingly, when phenyltins are added to macrophages in culture, they inflict massive alterations of cell morphology and interfere with membrane-associated processes, as visualized using fluorescence labelling of selected subcellular compartments.

Inflammasome Activation by ATP Enhances Citrobacter rodentium Clearance through ROS Generation.

BACKGROUND: Nod-like receptor family, pyrin domain containing 3 (NLRP3) is an important cytosolic sensor of cellular stress and infection. Once activated, NLRP3 forms a multiprotein complex (inflammasome) that triggers the maturation and secretion of interleukin (IL)-1ß and IL-18. We aimed to define the consequences of NLRP3 induction, utilizing exogenous adenosine triphosphate (ATP) as an inflammasome activator, to determine if inflammasome activation increases macrophage killing of Citrobacter rodentium and define mechanisms. METHODS: Bacterial survival was measured using a gentamicin protection assay. Inflammasome activation or inhibition in mouse J774A.1 macrophages were assessed by measuring IL-1ß; cytokines and reactive oxygen species (ROS) were measured by ELISA and DCFDA, respectively. RESULTS: Activation of the inflammasome increased bacterial killing by macrophages and its inhibition attenuated this effect with no impact on phagocytosis or cell death. Furthermore, inflammasome activation suppressed pro-inflammatory cytokines during infection, possibly due to more effective bacterial killing. While the infection increased ROS production, this effect was reduced by inflammasome inhibitors, indicating that ROS is inflammasome-dependent. ROS inhibitors increased bacterial survival in the presence of ATP, suggesting that inflammasome-induced bacterial killing is mediated, at least in part, by ROS activity. CONCLUSION: Improving inflammasome activity during infection may increase bacterial clearance by macrophages and reduce subsequent microbe-induced inflammation.

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.

Interaction and cellular uptake of surface-modified carbon dot nanoparticles by J774.1 macrophages.

Carbon dot (Cdot) nanoparticles are an emerging class of carbon nanomaterials with a promising potential for drug delivery and bio imaging applications. Although the interaction between Cdots and non-immune cell types has been well studied, Cdot interactions with macrophages have not been investigated. Exposure of Cdot nanoparticles to J774.1 cells, a murine macrophage cell line, resulted in minimal toxicity, where notable toxicity was only seen with Cdot concentrations higher than 0.5 mg/ml. Flow cytometric analysis revealed that Cdots prepared from citric acid were internalized at significantly higher levels by macrophages compared with those prepared from bamboo leaves. Interestingly, macrophages preferentially took up phenylboronic acid (PB)-modified nanoparticles. By fluorescence microscopy, strong blue light-specific punctate Cdot fluorescence resembling Cdot structures in the cytosolic space was mostly observed in J774.1 macrophages exposed to PB-modified nanoparticles and not unmodified Cdot nanoparticles. PB binds to sialic acid residues that are overexpressed on diseased cell surfaces. Our findings demonstrate that PB-conjugated Cdots can be taken up by macrophages with low toxicity and high efficiency. These modified Cdots can be used to deliver drugs to suppress or eliminate aberrant immune cells such as macrophages associated with tumors such as tumor-associated macrophages.

Chylomicron remnant model emulsions induce intracellular cholesterol accumulation and cell death due to lysosomal destabilization.

Chylomicron remnants, which carry dietary fats and cholesterol, play a role in promoting atherosclerosis. Chylomicron remnants are characterized by high cholesterol content at the surface, different from low-density lipoproteins (LDLs) containing high amounts of esterified cholesterol (CE) in the core. We prepared cholesterol-rich emulsions (TO-PC/cholesterol emulsions) as models for chylomicron remnants and compared their effects on J774 macrophages with acetylated-LDL (ac-LDL). Internalization of TO-PC/cholesterol emulsions into macrophages reduced cell viability, whereas ac-LDL did not. Surprisingly, there was no difference in intracellular free cholesterol content between cells incubated with TO-PC/cholesterol emulsions and with ac-LDL. Furthermore, cholesterol in TO-PC/cholesterol emulsions and ac-LDL both were internalized into J774 macrophages; however, incubation with TO-PC/cholesterol emulsions induced leakage of lysosomal protease, cathepsin-L, to cytosol, which was not observed for incubation with ac-LDL. Inhibition of the activity of cathepsin-L recovered the viability of macrophages that ingested TO-PC/cholesterol emulsions. We suggest an alternative fate of cholesterol-rich emulsions taken up by macrophages, which is different from other atherogenic lipoproteins rich in CE; internalization of TO-PC/cholesterol emulsions into macrophages induces rapid free cholesterol accumulation in lysosomes and cell death due to lysosomal destabilization.