Macrophage Cell Line J774 Research Articles

Pro-inflammatory Effects of Influenza Type A Virus PB1-F2 Protein-derived Peptide in Lipopolysaccharide-treated Macrophages.

Influenza A virus (IAV) has the potential to cause pandemics with considerable health and socio-economic burdens. A viral protein, polymerase basic 1- frame2 (PB1-F2), as a virulence factor, has pro-apoptotic activity and contributes to viral pathogenesis by delaying viral clearance and inducing inflammation. Macrophages are susceptible to IAV infection and produce high levels of inflammatory cytokines and chemokines. In the present study, the pro-inflammatory effects of PB1-F2 derived peptide was evaluated by measuring the expression of key inflammatory mediators in murine macrophage cell line J774.1. PB1-F2 treated macrophages were examined for nitric oxide (NO) production, inflammatory cytokines, and enzymes expression and pro-inflammatory cytokines secretion using Griess reagent, real-time polymerase chain reaction (PCR) and ELISA, respectively. Our results have shown that PB1-F2 peptide at non-cytotoxic concentrations (0.1-0.8 µmol/mL) had no effect on NO production. When applied to Lipopolysaccharide (LPS)-treated macrophages, PB1-F2 peptide at 0.8 μmol/mLincreasedinducible NO synthase (iNOS), cyclooxygenase (COX)-2, and interleukin (IL)-6 genes expression to 2.02, 3.81, and 3.65 folds, respectively. PB1-F2 at concentrations of 0.4 and 0.8 µm/mL increased tumor necrosis factor (TNF)-α transcription by 4.15 and 5.55 fold. At posttranslational level, TNF-α increased from 166.5±13.88 in LPS-treated cells to 773.6±95.27 and 1485±76.31 at concentrations of 0.4 and 0.8 μmol/mL in PB1-F2 peptide, respectively. However, PB1-F2 Peptide did not have any significant effect on IL-6 production. These findings suggest that PB1-F2 peptide may partly exert its enhancing role in viral pathogenicity through the induction of inflammatory mediators in macrophages. Hence, targeting PB1-F2 peptide would be helpful in the reduction of viral infection complications.

Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages.

Poly(D,L-lactide-co-glycolic) acid (PLGA) is a synthetic copolymer that has been used to design micro/nanoparticles as a carrier for macromolecules, such as protein and nucleic acids, that can be internalized by the endocytosis pathway. However, it is difficult to control the intracellular delivery to target organelles. Here we report an intracellular delivery system of nanoparticles modified with bacterial cytotoxins to the endoplasmic reticulum (ER) and anti-inflammatory activity of the nanoparticles. Subtilase cytotoxin (SubAB) is a bacterial toxin in certain enterohemorrhagic Escherichia coli (EHEC) strains that cleaves the host ER chaperone BiP and suppresses nuclear factor-kappaB (NF-κB) activation and nitric oxide (NO) generation in macrophages at sub-lethal concentration. PLGA-nanoparticles were modified with oligo histidine-tagged (6 × His-tagged) recombinant SubAB (SubAB-PLGA) through a pH-sensitive linkage, and their translocation to the ER in macrophage cell line J774.1 cells, effects on inducible NO synthase (iNOS), and levels of tumor necrosis factor (TNF)-α cytokine induced by lipopolysaccharide (LPS) were examined. Compared with free SubAB, SubAB-PLGA was significantly effective in BiP cleavage and the induction of the ER stress marker C/EBP homologous protein (CHOP) in J774.1 cells. Furthermore, SubAB-PLGA attenuated LPS-stimulated induction of iNOS and TNF-α. Our findings provide useful information for protein delivery to macrophages and may encourage therapeutic applications of nanoparticles to the treatment of inflammatory diseases.

Leishmanicidal Effects of Allium giganteum Saponin-Rich Fraction on Leishmania major

Background and objectives: Leishmaniasis is caused by the genus of Leishmania and is one of the important health problems worldwide. Serious side effects, the lack of effective vaccines and the emergence of drug resistance are the major weak points of leishmaniasis treatment. The purpose of this study was to evaluate leishmanicidal effects of Allium giganteum saponin rich fraction, natural compounds with history of antimicrobial properties, on promastigotes and axenic amastigotes of L. major and macrophages cell line J774. Methods: The chloroform-methanol (9:1) extract of the flowers was fractionated by MPLC using an RP-18 column. The saponin-rich fraction was detected by TLC and H-NMR analyses and evaluated for leishmanicidal activity on L. major and macrophages cell line J774 using MTT assay at 24, 48 and 72 h of incubation. Results: At concentrations of 75, 100 and 150 μg/mL, over the time of 24 to 72 h, a significant decrease in the live parasite's rate was observed (p <0.05). At 200 μg/mL concentration, all parasites were killed and maximum leishmanicidal effect was observed. The IC50s for promastigotes and axenic amastigotes were 90.01 ± 13.42 μg/mL and 29.76±17.91 μg/mL, respectively; the value for the J774 macrophage cell line was 33.17±4 μg/mL. Conclusion: The results of this study showed the significant leishmanicidal effect of saponin rich fraction from Allium giganteum on promastigote and axenic amastigote of L. major and macrophage cell line in vitro. Complementary in vivo studies for evaluating the effects of the fraction on leishmaniasis in BALB/c mice is recommended.

265-OR: Myeloid CREBZF Couples Inflammatory Signals to Systemic Insulin Resistance by Regulating Immune Response of the Adipose Tissue

Obesity and type 2 diabetes are associated with chronic inflammation response in the adipose tissue, in which macrophage activation and infiltration are hallmark characteristics. However, the molecular mechanism of this association remains largely unknown. Here, we show that CREB/ATF bZIP transcription factor CREBZF as a critical regulator of the adipose macrophage sensing inflammatory signals to cause systemic insulin resistance in obesity. We found that CREBZF was markedly induced in bone-marrow-derived macrophages and J774 macrophage cell lines in response to lipopolysaccharide treatment. To elucidate the function of CREBZF, we generated a myeloid cell-specific CREBZF knockout (CREBZF MKO) mouse by crossing floxed CREBZF mice with LysM-Cre transgenic mice. Myeloid cell-specific CREBZF deficiency in mice largely attenuated high-fat high sucrose (HFHS) diet-induced obesity and insulin resistance and hyperglycemia. CREBZF MKO mice showed a dramatic reduction of infiltrated macrophages in the white adipose tissue and an induction of whole-body energy expenditure. Ablation of CREBZF in myeloid cells caused a potent reduction of M1 macrophages and a concomitant induction of M2 macrophages in the adipose tissue of mice fed with HFHS diet, suggesting an important role of CREBZF in regulating adipose tissue macrophage polarization. Interestingly, CREBZF is capable of potentiating NF-κB signaling as evidenced by increased phosphorylation of IκB and p65 in the macrophage, whereas CREBZF deficiency resulted in opposite effects. These data indicate that CREBZF plays an important role in the regulation of innate immune response in the macrophage, and maintains whole-body energy metabolism and glucose homeostasis. Pharmacologic manipulation of CREBZF activity in the macrophage may provide novel opportunities for treating obesity, insulin resistance and type 2 diabetes. Disclosure Z. Hu: None. Y. Liu: None. Z. Liu: None. Z. Zhao: None. A. Cui: None. F. Ma: None. Y. Xue: None. Y. Han: None. F.F. Zhang: None. J. Fan: None. Y. Li: None. Funding National Key Research and Development Program of China (2017YFC0909601); National Natural Science Foundation of China (31471129, 31671224); Chinese Academy of Sciences (KJZD-EW-G20-02); K.C. Wong Education Foundation (to Y.L.)

Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

Lethal effects of gold nanoparticles on protoscolices of hydatid cyst: in vitro study

Recurrence of hydatidosis may results from cyst fluid spillage during surgery. To inactivate the cyst content, different scolicidal agents have been introduced. Nevertheless, novel and more effective treatments are needed due to the associated complications. In the current study, we examined the scolicidal effects of gold nanoparticles on Echinococcus granulosus protoscoleces. Hydatid cysts of sheep liver were collected in this study. The cyst fluid containing protoscoleces was aspirated aseptically. The protoscoleces were exposed to gold nanoparticles at different concentrations (250, 500, 1000, 2000, and 4000 μg/mL). Eosin staining method was applied to determine the viability of protoscoleces at 5, 10, 20, 30, and 60 min. Moreover, cytotoxicity analysis, scanning electron microscopy (SEM) of ultrastructural changes, DNA fragmentation, were performed, and protoscoleces were studied following treatment. The significant scolicidal effects of gold nanoparticles were observed at all concentrations compared to the control group. A total of 4000 μg/mL of gold nanoparticles could destroy 76% of protoscoleces in 60 min. Cytotoxic effects of gold nanoparticle on J774 macrophage cell line, in minimum and maximum concentration (500 and 4000 μg/mL), were 1 and 11%, respectively. After treatment of protoscoleces with gold nanoparticles in different times and concentrations, considerable alteration in size, ultrastructure changes in tegument and shape of sucker, as well as DNA fragmentation of parasite were seen. Based on the results, gold nanoparticles could exert scolicidal effects on hydatid cyst protoscoleces; therefore, they can be applied in hydatid cyst treatment.

Involvement of hepcidin in iron metabolism dysregulation in Gaucher disease.

Gaucher disease (GD) is an inherited deficiency of glucocerebrosidase leading to accumulation of glucosylceramide in tissues such as the spleen, liver, and bone marrow. The resulting lipid-laden macrophages lead to the appearance of “Gaucher cells”. Anemia associated with an unexplained hyperferritinemia is a frequent finding in GD, but whether this pathogenesis is related to an iron metabolism disorder has remained unclear. To investigate this issue, we explored the iron status of a large cohort of 90 type I GD patients, including 66 patients treated with enzyme replacement therapy. Ten of the patients treated with enzyme replacement were followed up before and during treatment. Serum levels of hepcidin, the iron regulatory peptide, remained within the physiological range, while the transferrin saturation was slightly decreased in children. Inflammation-independent hyperferritinemia was found in 65% of the patients, and Perl’s staining of the spleen and marrow smear revealed iron accumulation in Gaucher cells. Treated patients exhibited reduced hyperferritinemia, increased transferrin saturation and transiently increased systemic hepcidin. In addition, the hepcidin and ferritin correlation was markedly improved, and, in most patients, the hemoglobin level was normalized. To further explore eventual iron sequestration in macrophages, we produce a Gaucher cells model by treating the J774 macrophage cell line with a glucocerebrosidase inhibitor and showed induced local hepcidin and membrane retrieval of the iron exporter, ferroportin. These data reveal the involvement of Gaucher cells in abnormal iron sequestration, which may explain the mechanism of hyperferritinemia in GD patients. Local hepcidin-ferroportin interaction was involved in this pathogenesis.

Improving anti-trypanosomal activity of alkamides isolated from Achillea fragrantissima

In previous studies the aerial parts of Achillea fragrantissima were found to have substantial antileishmanial and antitrypanosomal activity. A bioassay-guided fractionation of a dichloromethane extract yielded the isolation of the essential anti-trypanosomal compounds of the plant. Seven sesquiterpene lactones (including Achillolide-A), two flavonoids, chrysosplenol-D and chrysosplenetine, and four alkamides (including pellitorine) were identified. This is the first report for the isolation of the sesquiterpene lactones 3 and 4, chrysosplenetine and the group of alkamides from this plant. Bioevaluation against Trypanosoma brucei brucei TC221 (T.b brucei) using the Alamar-Blue assay revealed the novel alkamide 13 to have an IC50 value of 40.37μM. A compound library, derived from the alkamide pellitorine (10), was synthesized and bioevaluated in order to find even more active substances. The most active compounds 26 and 27 showed activities in submicromolar concentrations and selectivity indices of 20.1 and 45.6, respectively, towards macrophage cell line J774.1. Toxicity of 26 and 27 was assessed using the greater wax moth Galleria mellonella larvae as an in vivo model. No significant toxicity was observed for the concentration range of 1.25–20mM.

Efficacy of Bioactive Cyclic Peptides in Rheumatoid Arthritis: Translation from In Vitro to In Vivo Models.

Using a novel drug discovery technology reported in previous issues of this journal cyclic peptides have been created which are able to down-regulate secretion of inflammatory cytokines, in vitro, by stimulated cells of the macrophage cell line J774. The cytokines in question, TNF-alpha and IL-6, are strongly implicated in etiology of diseases such as rheumatoid arthritis. Studies are reported here using the CAIA animal model for rheumatoid arthritis, which show that the peptides identified are indeed able to impact on inflammation of joints, induced in vivo. The results suggest that these peptides are effective at a dose which could be viable in man, and at which no adverse side effects are evident in the short term.

Measurement of the Phagocytic Activity of Human Peripheral Blood Using a Highly Sensitive Fluorometric Detection Device Without Hemolysis.

Phagocytes recognize pathogens that enter the body as well as other abnormal and foreign materials that may exist within an organism (such as dead cells, oxidized lipids, and denatured proteins), and phagocytose and eliminate them to maintain a healthy state. In a previous study a simple prototype device was used, under development by Hamamatsu Photonics (Prototype), that detects fluorescence to determine the phagocytic activity of the murine macrophage cell line J774.1. The present study aimed to determine whether it was possible to detect phagocytic activity in a slight amount of human peripheral blood without using hemolysis. Three microliters of human peripheral blood was drawn from the fingertip and mixed with 30 μg of pH-sensitive fluorescent particles. The fluorescence intensity of the human peripheral blood sample was then measured using the Prototype in development, cultured for 2 h at 37°C, and then re-measured. The phagocytes were observed under fluorescence microscopy and the phagocytosis rate of CD11b-positive cells was verified with a flow cytometer. The phagocytic activity of non-hemolyzed human peripheral blood was measured using the Prototype under development; fluorescence after phagocytosis was detected. Furthermore, this was confirmed by both fluorescence microscopy and flow cytometry. The precision of the measurements of human peripheral blood phagocytic activity was verified with the Prototype using samples from three healthy individuals. The relationship between blood sugar levels and phagocytic activity before and after meal times was determined. Concerning exercise, phagocytic activity tended to decrease, although salivary amylase level increased in the healthy individual examined after exercise. The simple Prototype can measure phagocytic activity in a small amount of peripheral blood without hemolysis. The device allows for rapid and minimally-invasive detection of changes in phagocytic activity, which has conventionally been difficult. These findings provide promising evidence that assessment of individual phagocytic capacity can be made easier using this novel device.

Abstract 73: Role of macrophages in the antitumor activity of an anti-transferrin receptor 1 antibody ch128.1 in a xenograft model of multiple myeloma

Abstract The transferrin receptor 1 (TfR1), also known as CD71, is a membrane glycoprotein involved in cellular iron uptake and regulation of cell growth. The high level of TfR1 expression on malignant cells and its key role in cancer cell pathology make this receptor an attractive target for antibody cancer therapy. We previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1). This antibody exhibits direct cytotoxicity against certain human malignant B cells in vitro through TfR1 degradation and iron deprivation. Importantly, ch128.1 shows remarkable anti-tumor activity in xenograft models of disseminated multiple myeloma (MM) in immunosuppressed mice (SCID-Beige). Interestingly, this anti-tumor protection was observed even against MM cells (KMS-11 cells) that show no sensitivity to this antibody in vitro, suggesting the in vivo contributions of antibody effector functions. This possibility was supported by the lack of anti-tumor protection observed using a ch128.1 Fc mutant with impaired binding to FcγRs and to the complement component C1q. To examine host effector functions involved in ch128.1-mediated protection in our mouse model bearing KMS-11 tumors, depletion studies of complement and macrophages were performed. Complement depletion using cobra venom factor (CVF) did not affect protection, suggesting that complement-mediated cytotoxicity (CDC) is not a relevant mechanism of action. Notably, we now report that macrophage depletion using clodronate liposomes (clodrolip) significantly reduced protection, suggesting that these effector cells play a relevant role in the anti-tumor activity. Consistent with this result, we also report that ch128.1 is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) against KMS-11 cells in the presence of the murine macrophage cell line J774.2 or murine bone marrow-derived macrophages. To examine the role of iron deprivation in ch128.1-mediated tumor death in vivo, animals treated with ch128.1 were systemically supplemented with iron in a preliminary study. However, no difference in survival was observed, suggesting that iron deprivation is not a contributor to the effects of ch128.1 in our in vivo model or the iron dose tested was not optimal. Our results suggest that macrophages play a key role in ch128.1-mediated anti-tumor protection in our model and that ch128.1 can be an effective therapy of incurable human B-cell malignancies such as MM. Citation Format: Lai Sum Leoh, Yoon Kyung Kim, Pierre V. Candelaria, Otoniel Martínez-Maza, Tracy R. Daniels-Wells, Manuel L. Penichet. Role of macrophages in the antitumor activity of an anti-transferrin receptor 1 antibody ch128.1 in a xenograft model of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 73. doi:10.1158/1538-7445.AM2017-73

Macrophage Migration Is Impaired within Candida albicans Biofilms.

Candida albicans is an opportunistic fungal pathogen that infects immunocompromised patients. Infection control requires phagocytosis by innate immune cells, including macrophages. Migration towards, and subsequent recognition of, C. albicans fungal cell wall components by macrophages is critical for phagocytosis. Using live-cell imaging of phagocytosis, the macrophage cell line J774.1 showed enhanced movement in response to C. albicans cell wall mutants, particularly during the first 30 min, irrespective of the infection ratio. However, phagocyte migration was reduced up to 2-fold within a C. albicans biofilm compared to planktonic fungal cells. Biofilms formed from C. albicans glycosylation mutant cells also inhibited macrophage migration to a similar extent as wildtype Candida biofilms, suggesting that the physical structure of the biofilm, rather than polysaccharide matrix composition, may hamper phagocyte migration. These data illustrate differential macrophage migratory capacities, dependent upon the form of C. albicans encountered. Impaired migration of macrophages within a C. albicans biofilm may contribute to the recalcitrant nature of clinical infections in which biofilm formation occurs.

Synthesis and biological evaluation of 2-methyl-1H-benzimidazole-5-carbohydrazides derivatives as modifiers of redox homeostasis of Trypanosoma cruzi

Twelve novel benzimidazole derivatives were synthesized and their in vitro activities against epimastigotes of Trypanosoma cruzi were evaluated. Two derivatives (6 and 7), which have 4-hydroxy-3-methoxyphenyl moiety in their structures, proved to be the most active in inhibiting the parasite growth. Compound 6 showed a trypanocidal activity higher than benznidazole (IC50=5µM and 7.5µM, respectively) and less than nifurtimox (IC50=3.6µM). In addition, the ability of 6 and 7 to modify the redox homeostasis in T cruzi epimastigote was studied; cysteine and glutathione increased in parasites exposed to both compounds, whereas trypanothione only increased with 7 treatment. These results suggest that the decrease in viability of T. cruzi may be attributed to the change in cellular redox balance caused by compound 6 or 7. Furthermore, compounds 6 and 7 showed CC50 values of 160.64 and 160.66µM when tested in mouse macrophage cell line J774 and selectivity indexes (macrophage/parasite) of 32 and 20.1, respectively.

RNA and a cell wall component of Enterococcus faecalis IC-1 are required for phagocytosis and interleukin 12 production by the mouse macrophage cell line J774.1

Enterococcus faecalis is a resident lactic acid bacterium in the human intestine. Its immunostimulatory action was reported to be enhanced by heat sterilization. To investigate its beneficial actions, we evaluated the ability of 10 E. faecalis strains to induce interleukin-12 (IL-12) production in a mouse macrophage cell line, J774.1 and found that the strain, E. faecalis IC-1, had a potent IL-12-inducing ability. Furthermore, we investigated the underlying mechanism by treating IC-1 cells with RNase or lysozyme. Its activity almost disappeared and an antagonist of Toll-like receptor (TLR) 7 inhibited this activity. Moreover, lysozyme-treated IC-1 bacteria were not phagocytized by J774.1 cells, and did not induce IL-12 production. Based on our results, we propose that macrophages recognize the cell wall components of IC-1, leading to phagocytosis. The IC-1 RNA is then recognized by TLR7, which induces the production of IL-12.

Promising antileishmanial effectiveness of doxorubicin and Doxil against Leishmania major : An in vitro assay

To evaluate the effect of doxorubicin and its pegylated liposomal formulation (Doxil, Caelyx) on invitro susceptibility of promastigote and amastigote stages of Leishmania major. Throughout invitro assays the IC50 was calculated in the promastigotes and amastigotes forms in J774 macrophage cell line. Also as cytotoxicity in J774cell line macrophages. Doxorubicin and Doxil showed the same activity against promastigote form with IC50 values of 10.49μg/mL and 9.63μg/mL, respectively. Similarly, the amastigote stage was susceptible at concentration of at least 1μg/mL when compared to positive control (P<0.0001). Also, cytotoxicity assay against macrophage revealed no toxicity on the host cells at IC50 concentrations. Our findings demonstrated the efficacy of both doxorubicin and its pegylated liposomal formulation on L. major at low concentrations. Further researches are needed for evaluating the safety of drugs in animal model particularly as topical formulation.

Production and characterization of functional recombinant hybrid heteropolymers of camel hepcidin and human ferritin H and L chains.

Hepcidin is a liver-synthesized hormone that plays a central role in the regulation of systemic iron homeostasis. To produce a new tool for its functional properties the cDNA coding for camel hepcidin-25 was cloned at the 5'end of human FTH sequence into the pASK-IBA43plus vector for expression in Escherichia coli The recombinant fusion hepcidin-ferritin-H subunit was isolated as an insoluble iron-containing protein. When alone it did not refold in a 24-mer ferritin molecule, but it did when renatured together with H- or L-ferritin chains. We obtained stable ferritin shells exposing about 4 hepcidin peptides per 24-mer shell. The molecules were then reduced and re-oxidized in a controlled manner to allow the formation of the proper hepcidin disulfide bridges. The functionality of the exposed hepcidin was confirmed by its ability to specifically bind the mouse macrophage cell line J774 that express ferroportin and to promote ferroportin degradation. This chimeric protein may be useful for studying the hepcidin-ferroportin interaction in cells and also as drug-delivery agent.

Dihydrotestosterone inhibits foam cell formation via a lectin-like ox-low-density lipoprotein receptor mediated mechanism in J774.1 cell line

Objective: To investigate the effect of dihydrotestosterone (DHT) on lectin-like ox- low-density lipoprotein (LDL) receptor(LOX-1)expression and foam cell formation in the female macrophage cell line J774.1. Methods: In cultured J774.1 cells, after pretreated with DHT at concentrations of 1×10-9 mol/L and 1×10-8 mol/L, ox-LDL-induced LOX-1 expression and foam cell formation were investigated by quantitative real-time PCR, Western blotting, and oil-red O staining. Results: DHT at concentrations of 1×10-9 mol/L and 1×10-8 mol/L inhibited ox-LDL-induced LOX-1 mRNA (2.81±0.46 and 2.29±0.21 vs 4.71±0.31, both P<0.01) and protein expression (1.35±0.06 and 1.09±0.04 vs 1.75±0.11, both P<0.05). The effect was partly reversed by the androgen receptor (AR) blocker flutamide (87.6%, P=0.004). Oil-red O staining also revealed that DHT at concentrations of 1×10-9 mol/L and 1×10-8 mol/L suppressed ox-LDL-induced foam cell formation as quantified by the number of foam cells per high-power field (HPF) (36.0±3.0 and 29.1±1.3 vs 45.9±3.7, both P<0.05) and by the area of oil-red O stained particles per HPF (7 983±1 035 and 4 060±390 vs 14 750±2 489, both P<0.05). Conclusion: DHT at concentrations of 1×10-9 mol/L and 1×10-8 mol/L decreases LOX-1 expression and foam cell formation via AR.

Dextran loading protects macrophages from lipid peroxidation and induces a Keap1/Nrf2/ARE-dependent antioxidant response

AimsLinear dextrans are often proposed as drug delivery systems with milder adverse effects and lower effective drug concentrations. Linear dextrans are polysaccharides that can potentially be used to load macrophages with drugs to transport them to a site of inflammation. Recently, it was reported that dextrans may exert a protective effect vis-à-vis drug cytotoxicity and during wound healing. The aim of the current work was to evaluate molecular mechanisms of action of dextrans that may be relevant to the cytoprotective effects. Main methodsWe determined the effect of treatment with 40- or 70-kDa dextran on production of reactive oxygen species, lipid peroxidation, and lysosomal pH in the J774 macrophage cell line. In addition, induction of Keap1/Nrf2/ARE and autophagic activity were evaluated. Key findingsDextrans of both molecular weights protected the cells from oxidative stress induced by cumene hydroperoxide and from lysosomal stress induced by ammonium chloride. The effect was associated with induction of the Keap1/Nrf2/ARE signaling pathway. Furthermore, dextran stimulated autophagy in a dose-dependent manner but inhibited the autophagosome-lysosome fusion in a time-dependent manner. SignificanceThis study shows possible cytoprotective effects of dextran under oxidative stress, and these findings may be used for the development of novel (dextran-based) drug delivery approaches.

Anti-inflammatory Effects of Nortrachelogenin in Murine J774 Macrophages and in Carrageenan-Induced Paw Edema Model in the Mouse.

Nortrachelogenin is a pharmacologically active lignan found in knot extracts of Pinus sylvestris. In previous studies, some lignans have been shown to have anti-inflammatory properties, which made nortrachelogenin an interesting candidate for our study. In inflammation, bacterial products and cytokines induce the expression of inducible nitric oxide synthase, cyclooxygenase-2, and microsomal prostaglandin E synthase-1. These enzymes synthesize factors, which, together with proinflammatory cytokines, are important mediators and drug targets in inflammatory diseases.The effects of nortrachelogenin on the expression of inducible nitric oxide synthase, cyclooxygenase-2, and microsomal prostaglandin E synthase-1 as well as on the production of nitric oxide, prostaglandin E2, and cytokines interleukin-6 and monocyte chemotactic protein-1 were investigated in the murine J774 macrophage cell line. In addition, we examined the effect of nortrachelogenin on carrageenan-induced paw inflammation in mice.Interestingly, nortrachelogenin reduced carrageenan-induced paw inflammation in mice and inhibited the production of inflammatory factors nitric oxide, prostaglandin E2, interleukin-6, and monocyte chemotactic protein-1 in J774 macrophages in vitro. Nortrachelogenin inhibited microsomal prostaglandin E synthase-1 protein expression but had no effect on cyclooxygenase-2 protein levels. Nortrachelogenin also had a clear inhibitory effect on inducible nitric oxide synthase protein expression but none on its mRNA levels, and the proteasome inhibitor lactacystin reversed the effect of nortrachelogenin on inducible nitric oxide synthase expression, suggesting a post-transcriptional mechanism of action.The results revealed hitherto unknown anti-inflammatory properties of nortrachelogenin, which could be utilized in the development of anti-inflammatory treatments.

Phagocytosis of bone marrow derived macrophages is controlled by phosphoinositide 3-kinase γ

Due to their ability to phagocytise invading microbes macrophages play a key role in the innate and acquired immune system. In this article the role of phosphoinositide 3-kinase gamma (PI3Kγ) for phagocytosis was studied in bone marrow derived macrophages (BMDM). By using genetic and pharmacological approaches our data clearly demonstrate PI3Kγ is acting as a mediator of macrophage phagocytosis. Phagocytosis of LPS activated BMDM was reduced in PI3Kγ depleted primary BMDM or macrophage cell line J774. Depletion of other class I phosphoinositide 3-kinases did not alter phagocytic activity. Partial reduction of the phagocytic index of BMDM expressing kinase inactive PI3Kγ indicate a lipid-kinase independent role of the PI3Kγ protein. Since inhibition of PI3Kγ interaction partner phosphodiesterase PDE3B reduced BMDM phagocytosis and PI3Kγ knock out super stimulated cAMP level, our data reveal that PI3Kγ protein mediated suppression of cAMP signalling is a critical for efficient phagocytosis of macrophages.