Inhibition of nitric oxide production of activated mice peritoneal macrophages is independent of the Toxoplasma gondii strain

BACKGROUND Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-β signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains. OBJECTIVES To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole. METHODS Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence. FINDINGS Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole. MAIN CONCLUSIONS These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.

Review of hypothesized mechanism of action of beta-glucan’s on cancer cells / Монголын Анагаах Ухаан

@#The human immune system has a remarkable ability to distinguish between the body’s own cells, recognized as “self” and foreign cells, as “nonself”, to mediate the immune responses. Mushroom β-glucans are a large group of macromolecules that are not naturally synthesized inside the human body, so these compounds are recognized as non-self-molecules, which activate the immunity. Mushroom polysaccharides or β-glucans are thought to provide their anti-tumor action primarily through the activation of the immune response of the host organism. In most cases mushroom polysaccharides do not directly affect tumor cells. </br> Certain β-glucans from medicinal mushrooms appear to activate cell-mediated and humoral immunity via activation of different immune cells, leading to elimination of tumor cells or pathogens. The activated macrophages (containing β-glucans) preferentially attack dead cells and intracellular pathogens. These macrophages also produce cytokines that prime natural killer cells and T lymphocytes, both of which are cytotoxic to tumor cells, via different mechanisms. Natural killer cells secrete chemical substances that destroy tumor cells by bursting cell membranes. Neutrophils effectively destroy targeted cells by cell mediated phagocytosis. In this review were described the hypothesized mechanism of action for fungal β-glucans against cancer cells.

Effect of Yiqi Yangyin Zhuyu Recipe in Anti-inflammation Activities in Vitro on LPS-induced RAW 264.7 Cells / 中国实验方剂学杂志

Objective: To investigate the effect of Yiqi Yangyin Zhuyu recipe on expressions of inflammatory factor and transformation of classically activated macrophages(M1)/alternatively activated macrophages (M2) inflammatory phenotype in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Method: Methyl-thiazdyl-tetrazolium(MTT) reduction assay was used to detect the effect of different concentrations of Yiqi Yangyin Zhuyu recipe on the proliferation of the cells. The release of nitric oxide was detected by the Griess method. Enzyme linked immunosorbent assay(ELISA) was used to detect the release of M1/M2 inflammatory cytokines in cell supernatant. The expressions of the pro-inflammatory factor genes of M1-macrophages and the anti-inflammatory factor genes of M2-macrophages were detected by Real-time PCR. The protein expression levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-1,nitric oxide synthase(iNOS) were detected by Western blot. Result: Results of MTT showed that Yiqi Yangyin Zhuyu recipe with the concentration of 2.0 g·L-1 and below had no effect on the cell proliferation. Results of Griess indicated that compared with blank group, the release of nitric oxide of LPS-induced group was increased (PPPPPPPα,IL-6,IL-1β,iNOS were up-regulated (Pα,IL-6,IL-1β,iNOS were down-regulated in Yiqi Yangyin Zhuyu recipe group, especially at the concentration at 2.0 g·L-1 (PConclusion: Yiqi Yangyin Zhuyu recipe could effectively inhibit the inflammatory reaction induced by LPS. The anti-inflammatory mechanism of Yiqi Yangyin Zhuyu recipe may be related to inhibition of macrophages to M1 phenotype polarization, so as to play the role of regulating immune and reducing the release of inflammatory cytokines, like NO,TNF-α,IL-6,IL-1β.

Protective effects of chronic clonorchis sinensis infestation on lungs of septic rat via M2 macrophages polarization / 中华急诊医学杂志

Objective To preliminarily investigate the protective effect of chronic clonorchis sinesis(Cs) infestation against sepsis in Sprague Dawley(SD) rats in order to explore its underlying mechanism.Methods Chronic Cs infestation model of SD rats was reproduced by intra-gastric administration with Cs ova.Twenty rats were randomly(random number) divided into normal group(n=10) and Cs group(n=10).The proportion of differentiation in M1 and M2 macrophages were detected by flow cytometry.The expressions of Arg-1(arginine-1),FIZZ 1,iNOs and TNF-αmRNA were examined by reverse transcriptase polymerase chain reaction(RT-PCR).The cecal ligation and puncture(CLP) procedure was performed to reproduce sepsis model of SD rats.Sixty rats were randomly(random number) divided into control group,SHAM group,CLP group,Mφ+CLP group,Cs-Mφ+CLP group,and Cs-CLP group.The cumulative mortalities were calculated.The pathological changes of the lung tissue in different groups were demonstrated by HE staining.The serum levels of cytokines TNF-α and IL-10 were detected by ELISA at 0,24,48 and 72 h after CLP procedure.Results Compared with M1 peritoneal macrophages differentiation in control group(91.9%),rat peritoneal macrophages were activated to M2 differentiation(95.1%) in chronic Cs infection group.RT-PCR assay showed expression of Arg-1 and FIZZ 1 mRNA were higher in M2 macrophages,and on the contrary, the expression of iNOS mRNA expression was higher in M1 macrophages.The expression of TNF-α mRNA in M1 was significantly higher than that in M2, whereas the expression of IL-10 mRNA in M2 was higher than that in M1.The cumulative mortality of septic rats 72 h after CLP procedure were much lower in both chronic Cs infestation group and M2 macrophages adoptive transfer group(CLP group 70%vs.Mφ+CLP group 50%vs.Cs-Mφ+CLP group 30%vs.Cs-CLP group 0%,P<0.05).In these two groups,the pathological damages in lung tissues were significantly improved.The serum level of TNF-α was decreased and the anti-inflammatory IL-10 level was increased significantly in these two groups with Cs compared with other groups.Conclusion M2 macrophages polarization induced by chronic Cs infestation with M2 phenotype gene and expression of anti-inflammatory cytokine gene play key role in increasing anti-inflammatory cytokines and decreasing pro-inflammatory cytokines to allerviate organ damage and ameliorating the survival rate in septic rats.

IL-4 Activated Macrophages Promote Hela Cell Migration and Invasion by Producing CCL18 / 中山大学学报(医学科学版)

Objective]To investigate the roles of IL-4 activated macrophages on Hela cells migration and invasion and the underlying mechanism.[Methods]The phenotype of IL-4 activated macrophage was assayed by morphology ,flow cytometry and Elisa. The ability of Hela cell migration and invasion was evaluated by transwell assay. The signaling proteins expression was displayed by Western blot.[Results]Il-4 activated macrophage had the phenotype of CD206high/HLA-DRlow ,and highly expressed IL-10 and CCL18 ,and decreased the expression of IL-12. The activated macrophages significantly promoted the migration and invasion of Hela cells ,while the unactivated macrophages did not had this effect. Applying anti-CCL18 antibody but not isotype IgG in the supernatant of IL-4 activated macrophage inhibited Hela cells motility. Furthermore,activated macrophages phosphorylated IKK and IκB and induced the translocation of p65 to nucleus ,eventually increased the expression of vimentin. Treatment of Hela cells with CCL18 also enhanced tumor cells migration and invasion ,as well as the NF-κB activation and vimentin expression.[Conclusion]IL-4 activated macrophages promoted Hela cells motility by secreting CCL18 and the following activation of NF-κB signaling proteins.

Paeoniflorin inhibits macrophage-mediated lung cancer metastasis / 中国天然药物

Alternatively activated macrophages are more frequently involved in tumor growth, angiogenesis, and immunosuppression. A previous study showed that paeoniflorin, the major active constituent of Paeonia lactiflora Pallas, can inhibit tumor growth and lung metastases of Lewis lung tumor-bearing mice. This study tried to investigate whether paeoniflorin inhibited lung cancer metastasis by inhibiting the alternative activation of macrophages (M2 macrophage). Using a viability assay, the cytotoxicity of paeoniflorin on Lewis lung cancer cells and peritoneal macrophages were investigated. In vitro scratch wound and in vivo lung metastasis experiments were used to test the ability to inhibit the migration of paeoniflorin and the function of M2 macrophages. Flow cytometry was performed to test the cell cycle of Lewis lung cancer cells, and to test the M2 macrophages in peritoneal macrophages and subcutaneous transplantable tumor. It was found that paeoniflorin showed no inhibitory effect on the growth of Lewis lung cancer cells and peritoneal macrophages of mouse in vitro. Paeoniflorin could attenuate the migration of LLC stimulated by alternatively activated macrophages (stimulated for 24 h and 48 h, paeoniflorin 1, 3, 10, 30, 100 μmol·L(-1), P < 0.01 or P < 0.05 vs control group). Paeoniflorin could decrease the cell populations at S phases (paeoniflorin 10, 30, 100 μmol·L(-1), P < 0.05 vs control group) and increase the cell populations at G0-G1 phases of Lewis lung cancer cells (paeoniflorin 100 μmol·L(-1), P < 0.05 vs control group) and reduce the numbers of M2 macrophages in peritoneal macrophages induced by IL-4 (paeoniflorin 1, 3, 10, 30, 100 μmol·L(-1), P < 0.01 vs Control group). Paeoniflorin could reduce lung metastasis of Lewis lung cancer cells xenograft and decrease the numbers of M2 macrophages in subcutaneous xenograft tumour in vivo (paeoniflorin 20, 40 mg·kg(-1), P < 0.01 vs control group). These results suggest that paeoniflorin could reduce lung metastasis of Lewis lung cancer cells xenograft partly through inhibiting the alternative activation of macrophages.

Efecto de cuatro ecotipos de Lepidium peruvianum Chacón sobre la producción de óxido nítrico in vitro

Los macrófagos desempeñan un rol importante en la respuesta innata y adaptativa, durante su activación producen mediadores citotóxicos como Óxido Nítrico (NO). El objetivo fue evaluar la producción de NO por macrófagos peritoneales de ratón cultivados con extractos metanólicos (EM) de los ecotipos rojo, negro, morado y blanco de Lepidium peruvianum Chacón (también conocida como Lepidium meyenii Walp). Los EM se prepararon empleando maca pulverizada macerada en metanol (1:2) durante 10 días. Los macrófagos peritoneales se obtuvieron de ratones 3 días después de haberles inyectado 1 ml de Caldo Tioglicolato por vía intraperitoneal; se cultivaron por triplicado durante 18 h a 37 °C en medio RPMI 1640 suplementado con 10% de suero de bovino fetal. La dosis de EM fue de 800 μg/ml por ecotipo, se consideraron controles sin EM. La producción de NO se determinó por acumulación de nitrito en el medio y se evidenció con el reactivo de Peter Griess, las concentraciones de nitrito se calcularon en base a la curva estándar elaborada con NaNO2. Las concentraciones producidas de nitrito fueron de 7,45; 6,79; 5,76; 5,61 y 6,81 mM para los EM de los ecotipos morado, negro, blanco, rojo y control respectivamente. Los cuatro ecotipos indujeron la producción de NO, aunque con el ecotipo morado fue superior (p>0,05).

The macrophages play an important role in the innate and adaptative response. Upon appropriate activation, macrophages released a variety of cytotoxic mediators like the Nitric Oxide (NO). The objective was to evaluate the production of NO for murine peritoneal macrophages cultivated with methanol extracts (ME) of different ecotypes of Lepidium peruvianum Chacón (at present Lepidium meyenii Walp.) red, black, purple and white. The ME was prepared using maca powdered macerated in methanol (1:2) during 10 days. Peritoneal macrophages were obtained of mice 3 days after having injected them 1 ml of thioglycolate broth; they were cultivated for triplicate during 18 hrs at 37 °C in RPMI 1640 medium supplemented with 10% fetal calf serum. The dose of ME was of 800 μg/ml for ecotype, were considered controls without ME. The production of NO was assayed by nitrite accumulation in the supernatant culture and it was evidenced with Peter Griess's reagent, the nitrite concentrations were calculated based on the standard curve elaborated with NaNO2. The produced concentrations of nitrite were of 7,45; 6,79; 5,76; 5,61 and 6,81 mM for the ME purple, black, white, red ecotypes and control respectively, although with the purple ecotype it was superior (p> 0,05).