Inflammatory responses in RAW264.7 macrophages caused by mycobacteria isolated from moldy houses

Mycobacterial strains (nonpathogenic Mycobacterium terrae, potentially pathogenic Mycobacterium avium-complex and Mycobacterium scrofulaceum), isolated from a moldy building, were studied with respect to their ability to stimulate macrophages (RAW264.7) to produce inflammatory mediators, and to cause cytotoxicity. Reactive oxygen species (ROS) were measured by chemiluminescence, cytokines (TNF-, IL-6, IL-1, IL-10) immunochemically, nitric oxide (NO) by Griess-method, expression of inducible NO-synthase (iNOS) with Western Blot analysis and cytotoxicity with MTT-test. All the strains induced dose- and time-dependent production of NO, IL-6 and TNF- in macrophages, whereas IL-1 or IL-10 production was not detected. The production of ROS and cytotoxicity was increased with the highest doses. Interestingly, different strains had significant differences in their ability to induce these responses, M. terrae being the most potent and M. avium-complex the weakest one. These results indicate that both non- and potentially pathogenic strains of mycobacteria present in moldy buildings are capable of activating inflammatory mechanisms in macrophages.     

Induction of cytotoxicity and production of inflammatory mediators in raw264.7 macrophages by spores grown on six different plasterboards

Dampness and microbial growth in buildings are associated with respiratory symptoms in the occupants, but details of the phenomenon are not sufficiently understood. The current study examined the effects of growth conditions provided by six plasterboards on cytotoxicity and inflammatory potential of the spores of Streptomyces californicus, Penicillium spinulosum, Aspergillus versicolor, and Stachybotrys chartarum. The microbes were isolated from mold problem buildings and thereafter grown on six different plasterboards. The spores were harvested, applied to RAW264.7 macrophages (10(4), 10(5), 10(6) spores/10(6) cells), and evaluated 24 h after exposure for the ability to cause cytotoxicity and to stimulate production of nitric oxide (NO), interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6). The data indicate clear differences between spores of different microbes in their ability to induce the production of these inflammatory mediators and to cause cell death in macrophages. Also, for each microbe, the induction ability specifically depended on the brand of plasterboard. The spores of Streptomyces californicus collected from all plasterboards were the most potent at inducing NO and cytokine production. Cytotoxicity caused by P. spinulosum and Streptomyces californicus spores was consistent with NO, IL-1beta and IL-6 production induced by those microbes. However, the production of these inflammatory mediators by the spores of Stachybotrys chartarum was not parallel to their ability to cause cell death. The low productions of NO and cytokines were associated with high cytotoxicity caused by the spores of the A. versicolor. These data suggest that growth condition of microbes on different plasterboards affect the ability of microbial spores to induce inflammatory responses and cytotoxicity in macrophages.     

EFFECT OF LINER AND CORE MATERIALS OF PLASTERBOARD ON MICROBIAL GROWTH,SPORE-INDUCED INFLAMMATORY RESPONSES,AND CYTOTOXICITY IN MACROPHAGES

Microorganisms, when grown on wetted plasterboards, can produce bioactive compounds capable of inducing inflammatory and toxic reactions in mammalian cells. The paper liner of plasterboard is commonly regarded as the major substrate for microbial growth. In this study, we cultured Stachybotrys chartarum, Aspergillus versicolor, Penicillium spinulosum, and Streptomyces californicus on liners and cores of plasterboards in order to examine the role of these main plasterboard components on microbial growth and the resulting bioactivity, which was assessed as the ability of microbial spores to induce inflammatory responses and to evoke cytotoxicity in mouse macrophages. The microbes, isolated from mold problem buildings, were grown under saturated humidity conditions on wetted liners and cores of six different plasterboards. The spores were collected, applied to RAW264.7 macrophages at different doses, and evaluated 24 h after exposure for their ability to evoke cytotoxicity and to stimulate production of nitric oxide (NO), tumor necrosis factor alpha (TNFα), and interleukin-6 (IL-6). In general, microbial growth was better on the cores than on the liners. All of the studied microbes collected from cores induced a dose-dependent production of TNFα in macrophages. The TNFα production stimulated by spores of Stachybotrys, Aspergillus, and Streptomyces paralleled their cytotoxicity. Spores of Streptomyces and Aspergillus collected from liners were among the most potent inducers of NO and IL-6. Good growth of Stachybotrys on cores was associated with high cytotoxicity. Penicillium grew only on cores, but it did not induce major inflammatory mediator productions, nor was it significantly cytotoxic. These results indicate that previously reported microbial growth on plasterboards and spore-induced production of important inflammatory mediators and cell death in macrophages is not only due to the paper liner of plasterboard, but the core material also has a crucial role.     

Effect of liner and core materials of plasterboard on microbial growth,spore-induced inflammatory responses,and cytotoxicity in macrophages

Microorganisms, when grown on wetted plasterboards, can produce bioactive compounds capable of inducing inflammatory and toxic reactions in mammalian cells. The paper liner of plasterboard is commonly regarded as the major substrate for microbial growth. In this study, we cultured Stachybotrys chartarum, Aspergillus versicolor, Penicillium spinulosum, and Streptomyces californicus on liners and cores of plasterboards in order to examine the role of these main plasterboard components on microbial growth and the resulting bioactivity, which was assessed as the ability of microbial spores to induce inflammatory responses and to evoke cytotoxicity in mouse macrophages. The microbes, isolated from mold problem buildings, were grown under saturated humidity conditions on wetted liners and cores of six different plasterboards. The spores were collected, applied to RAW264.7 macrophages at different doses, and evaluated 24 h after exposure for their ability to evoke cytotoxicity and to stimulate production of nitric oxide (NO), tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6). In general, microbial growth was better on the cores than on the liners. All of the studied microbes collected from cores induced a dose-dependent production of TNFalpha in macrophages. The TNFalpha production stimulated by spores of Stachybotrys, Aspergillus, and Streptomyces paralleled their cytotoxicity. Spores of Streptomyces and Aspergillus collected from liners were among the most potent inducers of NO and IL-6. Good growth of Stachybotrys on cores was associated with high cytotoxicity. Penicillium grew only on cores, but it did not induce major inflammatory mediator productions, nor was it significantly cytotoxic. These results indicate that previously reported microbial growth on plasterboards and spore-induced production of important inflammatory mediators and cell death in macrophages is not only due to the paper liner of plasterboard, but the core material also has a crucial role.     

氧化还原蛋白1对RAW264.7巨噬细胞炎症反应的刺激作用

目的 研究过氧化还原蛋白1(PORD1)对RAW264.7细胞炎性介质一氧化氮(NO)和肿瘤坏死因子α(TNFα)释放量的影响.方法 用MTT法检测重组蛋白PORD1对RAW264.7细胞的毒性,一步法检测RAW264.7细胞上清液中NO的含量;ELISA法检测RAW264.7细胞上清中TNFα的含量.结果 与空白组比较,50 nmol· L-1PORD1对细胞有显著毒性,20、10、5 nmol·L-1均无细胞毒性;PORD1可以诱导RAW264.7细胞中NO和TNFα大量表达.结论 外源性重组蛋白PORD1可以促进炎症反应的发生,其机制可能与增强炎症介质的释放有关.     

COMPARISON OF MYCOBACTERIA-INDUCED CYTOTOXICITY AND INFLAMMATORY RESPONSES IN HUMAN AND MOUSE CELL LINES

Environmental mycobacteria, which are ubiquitous in nature, are also detected in moisture-damaged buildings. Their potential role inducing the adverse health effects associated with living in moisture damaged buildings requires clarification. To establish a model for these studies, we evaluated inflammatory responsiveness in different cell lines exposed to environmental mycobacterial species. Four mycobacterial isolates belonging to Mycobacterium avium complex and Mycobacterium terrae, recovered from the indoor air sampled when a moldy building was being demolished, were studied for their cytotoxicity and ability to stimulate the production of inflammatory mediators in mouse RAW264.7 and human 28SC macrophage cell lines, and human A549 lung epithelial cell line. Lipopolysaccharide (LPS) was used as a positive control. Production of cytokines (tumor necrosis factorα, TNF-α; interleukin 6, IL-6; and interleukinβ, IL-1β) was analyzed immunochemically, nitric oxide (NO) by the Griess method, expression of inducible NO synthase with Western blot analysis, and cytotoxicity with the MTT test. Both human and mouse cells produced NO and IL-6 after mycobacterial exposure. Mouse macrophages also showed production of TNF-α induced by both mycobacteria and LPS, whereas the human cell lines failed to produce TNF-α after mycobacterial exposure and the human epithelial cell line also failed to respond to LPS. Similarly, only mouse macrophages produced IL-1β. Mycobacterial exposure was not cytotoxic to human cells and was only slightly cytotoxic to mouse macrophages. The results indicate that environmental mycobacterial isolates from moldy buildings are capable of activating inflammatory mechanisms in both human and murine cells. The human and mouse cell lines, however, differ significantly in the grade and type of the responses.     

Diesel particles induce nitric oxide production in murine alveolar macrophages and rat airways

The acute adverse health effects among respiratory and cardiovascular patients have been associated with particulate air pollution, containing diesel particles (DP). The mechanisms of these effects are unknown, but they may involve inflammation. We investigated the effects of DP (30-3000 μg/10(6) cells) on cell viability and production of nitric oxide (NO), interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in murine RAW 264.7 macrophage cultures in vitro. DP caused a dose- and time-dependent NO-production and was cytotoxic in murine RAW 264.7 macrophages. Cytotoxicity preceded the increases in NO production. DP had minimal effects on cytokine production. A single intratracheal instillation of DP 1 and 5 mg/rat increased NO production and protein concentration in bronchoalveolar lavage fluid, and caused pulmonary edema and hemorrhage. The present results indicate that DP can induce both NO production and cytotoxicity in the lower respiratory tract, which may contribute to the short-term adverse respiratory effects of these particles.     

Chromene suppresses the activation of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 cells

Inflammation is complex process involving a variety of immune cells that defend the body from harmful stimuli. However, pro-inflammatory cytokines and inflammatory mediators can also exacerbate diseases such as cancer. The aim of this study was to identify a natural effective remedy for inflammation. We isolated a functional algal chromene compound from Sargassum siliquastrum, named sargachromanol D (SD). We evaluated the anti-inflammatory effect of SD on lipopolysaccharide (LPS)-exposed RAW 264.7 cells by measuring cell viability, cytotoxicity, and production of inflammatory mediators such as nitric oxide (NO), prostaglandin E₂ (PGE₂), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. SD inhibited production of NO and PGE₂ from LPS-induced cells by preventing the expression of inflammatory mediators such as iNOS and COX-2 in a dose-dependent manner. Concurrently, levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were reduced with increasing concentrations of SD. In addition, SD inhibited the activation of NF-κB and mitogen-activated protein kinases (MAPKs) pathways in a concentration-dependent manner. These results indicate that SD inhibits LPS-stimulated inflammation by inhibition of the NF-κB and MAPKs pathways in macrophages.     

Immunomodulatory activities of Gelidium amansii gel extracts on murine RAW 264.7 macrophages

The objective of this study was to investigate the effects of Gelidium amansii, a red algae cultivated in the northeastern coast of Taiwan, on immune regulatory activities in RAW 264.7 macrophages. G. amansii gel was prepared by boiling and filtering the dried G. amansii algae. Phosphate-buffered saline (PBS) and ethanol extracts of the freeze-dried gel were then used to treat the cells. The results showed that the PBS extracts of G. amansii gel activated the macrophage by increasing cell proliferation and by enhancing the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6; the ethanol extracts did not show such effects. By contrast, neither PBS nor ethanol extracts of G. amansii gel affected NO production in lipopolysaccharide (LPS)-stimulated macrophages, although the ethanol extracts suppressed LPS-stimulated TNF-α, IL-1β, and IL-6 production. In conclusion, the PBS extracts of G. amansii gel activated the macrophage by enhancing the production of immune mediators, whereas the ethanol extracts showed anti-inflammatory activities by suppressing the cytokine production in LPS-stimulated RAW 264.7 macrophages.     

The proportions of Streptomyces californicus and Stachybotrys chartarum in simultaneous exposure affect inflammatory responses in mouse RAW264.7 macrophages

Adverse health outcomes associated with moisture-damaged buildings originate from an exposure consisting of complex interactions between various microbial species and other indoor pollutants. The concentrations and proportions of microbial components in such environments can vary greatly with the growth conditions. In this study, we aimed to evaluate the effects of simultaneous exposure with modified proportions of actinobacteria Streptomyces californicus and fungi Stachybotrys chartarum on inflammatory responses (cytokines macrophage inflammatory protein 2 [MIP2], interleukin 6 [IL-6] and tumor necrosis factor a [TNFa]; nitric oxide) and cytotoxicity (MTT-test and DNA content analysis) in mouse RAW264.7 macrophage cell line. Five different proportions of microbial spores were studied (Str. californicus: S. chartarum 10:1; 5:1; 1:1; 1:5; 1:10). RAW264.7 cells were coexposed to the total dose of 3x10(5) spores/ml for 24 h and also both of these microbial spores on their own at the respective doses. At least the 1.5-fold synergistic increase in cytokine production of RAW264.7 macrophages was detected when coexposure contained an equal amount or more fungal spores (S. chartarum) than bacterial spores (Str. californicus) compared to the sum response caused by these microbial spores separately. On the contrary, NO production after coexposure was nearly 40% less than the sum response induced by the microbial spores separately, when coexposure contains 5 times more bacterial than fungal spores. In addition, coexposure slightly changed the cytotoxic potency of the spores. The present results revealed that mutual proportions of fungal and bacterial spores in simultaneous exposure affect the nature of their interactions leading to increased or suppressed production of inflammatory mediators in RAW264.7 macrophages.     

Isosteroid alkaloids with different chemical structures from Fritillariae cirrhosae bulbus alleviate LPS-induced inflammatory response in RAW 264.7 cells by MAPK signaling pathway

Isosteroid alkaloids, natural products from Fritillariae Cirrhosae Bulbus, are well known for its antitussive, expectorant, anti-asthmatic and anti-inflammatory properties. However, the anti-inflammatory effect and its mechanism have not been fully explored. In this study, the anti-inflammatory activitives and the potential mechanisms of five isosteroid alkaloids from F. Cirrhosae Bulbus were investigated in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. The pro-inflammatory mediators and cytokines were measured by Griess reagent, ELISA and qRT-PCR. The expression of MAPKs was investigated by western blotting. Treatment with the five isosteroid alkaloids in appropriate concentrations could reduce the production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in supernatant, and suppressed the mRNA expressions of TNF-α and IL-6. Meanwhile, the five isosteroid alkaloids significantly inhibited the phosphorylated activation of mitogen activated protein kinase (MAPK) signaling pathways, including extracellular signal-regulated kinase (ERK1/2), p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). These results demonstrated that isosteroid alkaloids from F. Cirrhosae Bulbus exert anti-inflammatory effects by down-regulating the level of inflammatory mediators via mediation of MAPK phosphorylation in LPS-induced RAW264.7 macrophages, thus could be candidates for the prevention and treatment of inflammatory diseases.     

Taurine chloramine inhibits NO and TNF-α production in zymosan plus interferon-γ activated RAW 264.7 cells

Taurine is present abundantly in various tissues, especially in leukocytes embattled to foreign invaders such as microorganisms or oxidants. Taurine-chloramine (Tau-Cl) is produced from taurine at the site of inflammation via the myeloperoxidase-halide pathway in leukocytes induced by oxidants and/or infectious materials. Previously, our data demonstrated that Tau-Cl inhibited nitric oxide (NO) production and TNF-α secretion induced by lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR-4) ligand or lipoarabinomannan (LAM), a TLR-2 ligand plus interferon-γ (IFN-γ) in peritoneal macrophages or RAW 264.7 cells. Zymosan, a β-glucan of yeast cell wall, is a ligand for TLR-2 and dectin-1 and stimulates macrophages to produce proinflammatory mediators such as NO and TNF-α. Based on our previous data, we examined the effect of zymosan and IFN-γ induced production of NO and TNF-α in the absence or presence of Tau-Cl or taurine using RAW 264.7 cells. Production of NO and secretion of TNF-α is increased when zymosan is combined with IFN-γ. Tau-Cl inhibited production of NO and secretion of TNF-α in zymosan plus IFN-γ activated RAW 264.7 cells in a dose-dependent manner (99% vs. 48% using 0.8mM Tau-Cl). Taurine was without effect. Nitric oxide synthase protein (iNOS), induced by zymosan plus IFN-γ, was inhibited by Tau-Cl (0.8mM) as measured using western blot analysis. NOS mRNA was inhibited by Tau-Cl at four, eight and 16 hours post activation, but not at 24 hours. TNF-α mRNA was inhibited at four hours and eight hours, but not at 16 and 24 hours. These data suggest that expression of both iNOS and TNF-α mRNAs are inhibited by treatment with Tau-Cl within four and eight hours, but not at later time points. Transient suppression of activation of RAW 264.7 cells induced by zymosan may play a critical physiological role for taurine in protecting against tissue injury from initial overt inflammation. This study indicates that tropical treatment of taurine may ameliorate inflammatory dermatoses caused by an environmental yeast or abnormal immune function.     

Britanin suppresses LPS-induced nitric oxide,PGE2 and cytokine production via NF-κB and MAPK inactivation in RAW 264.7 cells

Little is known about the biological properties of britanin, which is isolated from the flowers of Inula japonica (Inulae Flos). Based on our previous studies that Inulae Flos had anti-inflammation and anti-asthmatic activities, we tried to find the bioactive compounds from it. In this study, the anti-inflammatory effects of britanin on the inflammatory mediators as well as on nuclear factor (NF)-кB and mitogen-activated protein (MAP) kinase activation were evaluated in RAW 264.7 cells. Britanin inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE₂) along with the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In addition, britanin reduced the release of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. Furthermore, the phosphorylations of MAP kinases (p38 and JNK) in LPS-stimulated RAW 264.7 cells were suppressed by britanin. Moreover, britanin inhibited the NF-κB activation induced by LPS, which was associated with the abrogation of IκBα degradation and subsequent decreases in nuclear p65 levels. This study suggests that the anti-inflammatory activities of britanin might be attributed to the inhibition of iNOS and COX-2 and cytokine expression at least in part, through the attenuation of the phosphorylations of MAP kinases and NF-κB activation via IκBα degradation in macrophages. We conclude that britanin may have potential for the treatment of inflammatory diseases through the down-regulation of MAP kinases and NF-κB mediated activation of macrophages.     

Asperlin from the marine-derived fungus Aspergillus sp. SF-5044 exerts anti-inflammatory effects through heme oxygenase-1 expression in murine macrophages

Asperlin is a fungal metabolite isolated from Aspergillus sp. SF-5044. In the present study, we isolated asperlin from the marine-derived fungus Aspergillus sp. SF-5044 and demonstrated that it inhibited inducible nitric oxide synthase (iNOS) expression, reduced iNOS-derived NO, suppressed cyclooxygenase (COX)-2 expression, and reduced COX-derived prostaglandin (PG) E? production in lipopolysaccharide (LPS)-stimulated RAW264.7 and murine peritoneal macrophages. Similarly, asperlin reduced the production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. In addition, asperlin inhibited the phosphorylation and degradation of IκB-α, as well as the nuclear translocation of p65 caused by the stimulation of LPS in RAW264.7 macrophages. Furthermore, asperlin induced heme oxygenase (HO)-1 expression through nuclear translocation of nuclear factor E2-related factor 2 and increased HO activity in RAW264.7 macrophages. The effects of asperlin on the LPS-induced expression of iNOS and COX-2 and production of NO, PGE?, TNF-α, and IL-1β were partially reversed by a HO-1 inhibitor, tin protoporphyrin. These findings suggest that asperlin-induced HO-1 expression plays a role in the anti-inflammatory effects of asperlin in macrophages.     

8,8′-Bieckol,isolated from edible brown algae,exerts its anti-inflammatory effects through inhibition of NF-κB signaling and ROS production in LPS-stimulated macrophages

Ecklonia cava (E. cava) is an abundant brown alga that contains high levels of phlorotannins, which are unique marine polyphenolic compounds. It has been suggested that E. cava phlorotannins exert anti-inflammatory effects. However, the anti-inflammatory effects and underlying molecular mechanism exerted by 8,8′-bieckol isolated from E. cava have not been reported. Thus, in this study, we examined the anti-inflammatory effects of 8,8′-bieckol on lipopolysaccharide (LPS)-stimulated primary macrophages and RAW 264.7 macrophages. We found that 8,8′-bieckol suppressed key inflammatory mediator [i.e., nitric oxide (NO) and prostaglandin E₂ (PGE₂)] production in both primary and RAW 264.7 macrophages. 8,8′-Bieckol inhibited NO by suppressing LPS-induced expression of inducible nitric oxide synthase (iNOS) at the mRNA and protein levels in primary macrophages and RAW 264.7 cells. In addition, 8,8′-bieckol decreased the production and mRNA expression of the inflammatory cytokine interleukin-6 (IL-6), but not tumor necrosis factor (TNF)-α, in RAW 264.7 cells. Moreover, 8,8′-bieckol treatment diminished transactivation of nuclear factor-kappa B (NF-κB) and nuclear translocation of the NF-κB p65 subunit and suppressed LPS-induced intracellular reactive oxygen species (ROS) production in macrophages. Furthermore, 8,8′-bieckol markedly reduced mortality in LPS-induced septic mice. Taken together, these data indicate that the anti-inflammatory properties of 8,8′-bieckol are associated with the suppression of NO, PGE₂, and IL-6 via negative regulation of the NF-κB pathway and ROS production in LPS-stimulated RAW 264.7 cells. Moreover, 8,8′-bieckol protects mice from endotoxin shock.