Human Macrophage Cell Line U937 Research Articles

Transcriptional Modification and Potential Intracellular Signaling Mechanisms in Human Macrophages Primed by Interferon-γ

To explore the transcriptional gene expression profile up-regulated in human macrophages stimulated by interferon-γ (IFN-γ) and the underlying intracellular signaling mechanisms. RNA-seq was used to sequence and compare the differential gene expression profiles of human macrophage cell line U937 before and after IFN-γ stimulation, and the significantly up-regulated genes were screened out, which were verified by fluorescence-based real-time quantitative polymerase chain reaction (qPCR) in U937 and THP1 cell lines, respectively. JAK/STAT, MAPK/ERK and PI3K/AKT pathway inhibitors were added to simultaneously to the cultured U937 cells upon IFN-γ priming to detect their effects on the expressions of the up-regulated genes to explore the key regulatory mechanisms. RNA-seq and qPCR results showed that, the well-recognized chemokines CXCL9, CXCL10 and CXCL11, the APOL family including APOL1, APOL2, APOL3, APOL4, APOL6 and GBP family GBP1, GBP2, GBP3, GBP4 and GBP5 as well were significantly up-regulated in IFN-γ-stimulated U937 cells. JAK/STAT3 pathway inhibitor inhibited the upregulation of APOL1, APOL4, GBP1, GBP4 and GBP5 genes induced by IFN-γ, while MAPK/ERK pathway inhibitor inhibited the upregulation of CXCL10 gene. PI3K/AKT pathway inhibitor inhibited the upregulation of APOL1,APOL4, APOL6, GBP1 and GBP5 genes induced by IFN-γ, all three signal pathway inhibitors could inhibit the upregulation of CXCL9 gene, and none of them could inhibit the upregulation of APOL3 gene. Upon IFN-γ stimulation, some family molecules of APOL and GBP in macrophages are significantly up-regulated, and PI3K/AKT, JAK/STAT3 and MAPK/ERK pathways have positive regulation on their expressions, respectively.

Cyclooxygenase-2 Upregulated by Temozolomide in Glioblastoma Cells Is Shuttled In Extracellular Vesicles Modifying Recipient Cell Phenotype.

Temozolomide (TMZ) resistance is frequent in patients with glioblastoma (GBM), a tumor characterized by a marked inflammatory microenvironment. Recently, we reported that cyclooxygenase-2 (COX-2) is upregulated in TMZ-resistant GBM cells treated with high TMZ concentrations. Moreover, COX-2 activity inhibition significantly counteracted TMZ-resistance of GBM cells. Extracellular vesicles (EV) are considered crucial mediators in orchestrating GBM drug resistance by modulating the tumor microenvironment (TME) and affecting the surrounding recipient cell phenotype and behavior. This work aimed to verify whether TMZ, at low and clinically relevant doses (5-20 µM), could induce COX-2 overexpression in GBM cells (T98G and U87MG) and explore if secreted EV shuttled COX-2 to recipient cells. The effect of COX-2 inhibitors (COXIB), Celecoxib (CXB), or NS398, alone or TMZ-combined, was also investigated. Our results indicated that TMZ at clinically relevant doses upregulated COX-2 in GBM cells. COXIB treatment significantly counteracted TMZ-induced COX-2 expression, confirming the crucial role of the COX-2/PGE2 system in TMZ-resistance. The COXIB specificity was verified on U251MG, COX-2 null GBM cells. Western blotting of GBM-EV cells showed the COX-2 presence, with the same intracellular trend, increasing in EV derived from TMZ-treated cells and decreasing in those derived from COXIB+TMZ-treated cells. We then evaluated the effect of EV secreted by TMZ-treated cells on U937 and U251MG, used as recipient cells. In human macrophage cell line U937, the internalization of EV derived by TMZ-T98G cells led to a shift versus a pro-tumor M2-like phenotype. On the other hand, EV from TMZ-T98G induced a significant decrease in TMZ sensitivity in U251MG cells. Overall, our results, in confirming the crucial role played by COX-2 in TMZ-resistance, provide the first evidence of the presence and effective functional transfer of this enzyme through EV derived from GBM cells, with multiple potential consequences at the level of TME.

Role of ESAT-6 in pathogenicity of Beijing and non-Beijing Mycobacterium tuberculosis isolates

BackgroundMycobacterium tuberculosis Beijing genotype was associated with tuberculosis outbreaks and increased transmissibility. To understand the variation in virulence between Beijing and non-Beijing clinical isolates of M.tuberculosis genotypes, the esat-6 gene sequencing, and its expression was compared in the macrophage environment. Materials & methodsAmong 64 nonrepetitive, culture-positive M.tuberculosis, DNA extraction of 24 and 40 pure confirmed Beijing and non-Beijing isolates was accompanied by the boiling method. esat-6 gene PCR amplification and their sequencing were carried out by specific primers and its expression was performed on human macrophage cell line U937 after 6, 12, and 18 h of exposure to bacilli. The esat-6 mRNA transcription and expression in M. tuberculosis treated macrophage by Real-Time PCR and Western blot method. ResultsData analysis based on sequencing of the east-6 gene PCR product showed that this gene exists in all isolates and there are no changes or single nucleotide variation between the Beijing and non-Beijing isolates. In Beijing strains, the esat-6 expression was increased during the study times, but it was constant in non-Beijing isolates. esat-6 gene expression in Beijing isolates reached to about 44.9 times more than non-Beijing isolates after 18 h incubation on the macrophages cell line. Conclusionesat-6 is a conserved gene both in Beijing and non-Beijing isolates of M.tuberculosis. More expression of the east-6 gene in the macrophage model may indicate that this gene is likely to play a more important role in increasing the pathogenicity of Beijing strains.

Highly accurate and label-free discrimination of single cancer cell using a plasmonic oxide-based nanoprobe

The detection of cancer cells at the single-cell level enables many novel functionalities such as next-generation cancer prognosis and accurate cellular analysis. While surface-enhanced Raman spectroscopy (SERS) has been widely considered as an effective tool in a low-cost and label-free manner, however, it is challenging to discriminate single cancer cells with an accuracy above 90% mainly due to the poor biocompatibility of the noble-metal-based SERS agents. Here, we report a dual-functional nanoprobe based on dopant-driven plasmonic oxides, demonstrating a maximum accuracy above 90% in distinguishing single THP-1 cell from peripheral blood mononuclear cell (PBMC) and human embryonic kidney (HEK) 293 from human macrophage cell line U937 based on their SERS patterns. Furthermore, this nanoprobe can be triggered by the bio-redox response from individual cells towards stimuli, empowering another complementary colorimetric cell detection, approximately achieving the unity discrimination accuracy at a single-cell level. Our strategy could potentially enable the future accurate and low-cost detection of cancer cells from mixed cell samples.

The Japanese Herbal (Kampo) Medicine Hochuekkito Attenuates Lung Inflammation in Lung Emphysema.

Chronic obstructive pulmonary disease (COPD) is a systemic inflammatory disorder. It often causes weight loss, which is considered a poor prognostic factor. A Japanese herbal Kampo medicine, Hochuekkito (TJ-41), has been reported to prevent systemic inflammation and weight loss in COPD patients, but the underlying biological mechanisms remain unknown. In the present study, we investigated the role of TJ-41 in vivo using a mouse model of lung emphysema. We used lung epithelium-specific Taz conditional knockout mice (Taz CKO mice) as the lung emphysema model mimicking the chronic pulmonary inflammation in COPD. Acute inflammation was induced by intratracheal lipopolysaccharide administration, simulating COPD exacerbation. Mice were fed a diet containing 2% TJ-41 or a control diet. Taz CKO mice showed increased numbers of inflammatory cells in the bronchoalveolar lavage fluid compared to control mice. This effect was reduced by TJ-41 treatment. In the acute exacerbation model, TJ-41 mitigated the increased numbers of inflammatory cells in the bronchoalveolar lavage fluid and attenuated lung inflammation in histopathological studies. Additional in vitro experiments using the human macrophage cell line U-937 demonstrated that lipopolysaccharide-induced tumor necrosis factor-alpha expression was significantly downregulated by TJ-41. These results suggest that TJ-41 has anti-inflammatory effects in lung emphysema both in the chronic phase and during an acute exacerbation. In conclusion, our study sheds light on the anti-inflammatory effects of TJ-41 in lung emphysema. This establishes its potential as a new anti-inflammatory therapy and a preventive medicine for exacerbations during the long-time maintenance of COPD patients.

LncRNA MEG3 expression in sepsis and its effect on LPS-induced macrophage function

Sepsis is a dangerous disease that is caused by an overreaction of the body's immune system to infection and gradually spreads throughout the body. This experiment was carried out to explore the expression of LncRNA MEG3 in sepsis and its effect on LPS-induced macrophage function. Methods 60 sepsis patients admitted to our hospital from February 2017 to September 2018 were selected as the sepsis group, and 50 non-septic patients diagnosed and treated in our hospital during the same period were selected as the control group. qRT-PCR was used to detect the expression level of MEG3. ROC curve was used to analyze the diagnostic value of serum MEG3 in sepsis. The human macrophage cell line U937 was cultured in vitro and randomly divided into NC group, LPS group, LPS + pcDNA group, and LPS + pcDNA-MEG3 group. Flow cytometry was applied to detect the apoptosis rate. The levels of IL-1β and TNF-α were detected by ELISA. Western blot was used to detect the expression of Bax, Bcl-2 and NF-κB signaling pathway-related proteins p65 and p-p65. Results: The expression level of serum MEG3 in the sepsis group was significantly lower than that in the control group (P <0.05). ROC curve analysis showed that the AUC area was 0.856, the sensitivity was 0.700, and the specificity was 0.883. Compared with the NC group, the macrophage apoptosis rate in the LPS group was increased (P <0.05), the levels of Bax and p-p65 protein were significantly increased (P <0.05), and the level of Bcl-2 protein was decreased (P <0.05), the levels of IL-1β and TNF-α were increased (P <0.05). Compared with the LPS + pcDNA group, the apoptosis rate of the LPS + pcDNA-MEG3 group was significantly reduced (P <0.05), the levels of Bax and p-p65 protein were reduced (P <0.05), and the level of Bcl-2 protein was significantly increased (P <0.05), the levels of IL-1β and TNF-α were reduced (P <0.05). Conclusion: The low expression of LncRNA MEG3 in the serum of patients with sepsis can predict the occurrence of sepsis. Overexpression of MEG3 can inhibit LPS-induced macrophage apoptosis and secretion of inflammatory factors by inhibiting the activation of the NF-κB signaling pathway.

Decidual stromal cells maintain decidual macrophage homeostasis by secreting IL-24 in early pregnancy.

The state of self-renewal and self-maintain of decidual macrophages would be important for immune homeostasis at the maternal-fetal interface. The roles of interleukin (IL)-24 derived from decidual stromal cells (DSCs) on decidual macrophages have not been explored. IL-24 expression in DSCs was interfered by lentivirus, and the transcription levels of IL-24 in DSCs were verified by real time (RT)-PCR. The levels of IL-24 receptors were determined by flow cytometry assays. The effect of recombination human IL-24 (rhIL-24) on the differentiation and apoptosis of macrophages was analyzed by flow cytometry in vitro. The viability of macrophages was detected by Cell Counting Kit-8 assays. The growth of DSCs was not affected obviously only by IL-24 knockdown while the growth of knockdown DSCs was inhibited significantly after co-cultured with decidual macrophages. The levels of IL-24 receptors (IL-20R1 and IL-22R1) were moderately to highly expressed on decidual macrophages and human macrophage cell line U937. The differentiation of decidual macrophages treated by rhIL-24 or co-cultured with IL-24 knockdown DSCs was not affected. Both apoptosis and viability of U937 cells were promoted by rhIL-24. The ratio of Bcl-2/Bax was down-regulated and Ki-67 was up-regulated by IL-24 treatment. The expression of Bcl-2/Bax was up-regulated while Ki-67 was down-regulated in U937 cells after co-cultured by IL-24 knockdown DSCs. IL-24 secreted by DSCs promotes the renewal and homeostasis of decidual macrophages possibly via down-regulating the ratio of Bcl-2/Bax and up-regulating of the expression of Ki-67 in early pregnancy.

Downregulation of miR-199a-3p mediated by the CtBP2-HDAC1-FOXP3 transcriptional complex contributes to acute lung injury by targeting NLRP1.

Emerging evidence indicates that microRNAs (miRNAs) play fundamental roles in the pathogenesis of multiple diseases, including acute lung injury (ALI). Here, we discovered that miR-199a-3p was significantly downregulated in ALI lung tissues using a microarray analysis. In vitro lipopolysaccharide (LPS) treatment of the human epithelial cell line A549 and the human macrophage cell line U937 caused a decrease of miR-199a-3p. Mechanically, miR-199a-3p specifically bound to the 3'-untranslated region (3'-UTR) of NLRP1 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 1), a critical member of inflammasomes. Ectopic overexpression or downregulation of miR-199a-3p resulted in the repression or induction of NLRP1, respectively, thereby downregulating or activating its downstream events. Moreover, transcription factor FOXP3 (forkhead box P3) was able to specifically bind to the promoter of miR-199a-3p. Knockdown or overexpression of FOXP3 resulted in a decrease or induction miR-199a-3p expression, respectively. Using immunoprecipitation (IP), mass spectrometry and co-IP assays, we found that FOXP3 formed a transcriptional complex with HDAC1 (histone deacetylase 1) and CtBP2 (C-terminal-binding protein 2). Collectively, our results suggested that the CtBP2-HDAC1-FOXP3 transcriptional complex (CHFTC) could specifically bind to the promoter of miR-199a-3p and repress its expression. Downregulation of miR-199a-3p eliminated its inhibition of NLRP1, causing activation of NLRP1 and cleavage of pro-IL-1β and pro-IL-18 mediated by Caspase-1. The secretion of IL-1β and IL-18 further aggravated the inflammatory response and resulted in the occurrence of ALI.

IL-33/ST2 axis affects the polarization and efferocytosis of decidual macrophages in early pregnancy.

To explore whether IL-33/ST2 axis modulates the polarization and efferocytosis of decidual macrophages (dMφs). The phenotype characteristics of dMφs from both normal pregnant women and recurrent spontaneous abortion (RSA) patients were determined by real-time polymerase chain reaction (RT-PCR) and flow cytometry (FCM). Then, the efferocytosis and expression of IL-33 and its receptor (ST2) in dMφs were analyzed by FCM. Finally, the effects of sST2, a decoy receptor for IL-33 that inhibits the IL-33/ST2 signaling pathway, on the polarization and efferocytosis of dMφs and human macrophage cell line U937 were investigated. Compared with normal pregnancy, dMφs from RSA patients presented a M1 phenotype with high secretion of IL-33, whereas the expression of ST2 decreased. However, dMφs from RSA patients possessed a more powerful efferocytosis ability to clear the apoptotic decidual stromal cells (DSCs) compared with dMφs from normal pregnancy patients. Treatment with recombinant human sST2 led to the up-regulation of M1 bias and efferocytosis ability of both normal dMφs and U937. This study indicates that IL-33 secreted by dMφs promotes M2 bias at the feto-maternal interface, and as a result, RSA might attribute to the disturbance of IL-33/ST2 axis and the enhancement of efferocytosis of dMφs subsequently.

Mycobacterium tuberculosisSerine Protease Rv3668c Can Manipulate the Host–Pathogen Interaction via Erk-NF-κB Axis-Mediated Cytokine Differential Expression

Tuberculosis caused by Mycobacterium tuberculosis (MTB) remains a serious global public health concern. About one-third of the global population has been latently infected with this pathogen. MTB proteases are important virulence factors and involve in subverting the host immunity. MTB protease Rv3668c was implicated in drug action and dormancy by Gene Expression Omnibus data. To define the role of Rv3668c in pathogen-host interaction, we constructed recombinant strain Mycobacterium smegmatis-Rv3668c (Ms-Rv3668c). The resultant strains were used to challenge the human macrophage cell line U937. The cytokine levels and the survival of recombinants and macrophages were monitored. The results showed that recombinant Ms-Rv3668c specifically upregulated the secretion of proinflammatory cytokines TNF-α, IL-1β, and IL-6 and downregulated the secretion of anti-inflammatory cytokine IL-10 by U937 cells, consistent with the upregulated transcription of TNF-α and IL-1β. Rv3668c recombinants demonstrated prolonged survival within the U937 cells and accelerated the death of the host cells. Inhibitor experiments showed that the ERK-NF-κB axis was involved in the Rv3668c-triggered TNF-α and IL-1β changes. These results provided evidence for the engagement of Rv3668c in the interaction between Mycobacterium and host.

HMGB1 release by human liver L02 and HepG2 cells induced by lipopolysaccharide

Liver cells release the high mobility group box-1 (HMGB1) protein when exposed to lipopolysaccharides (LPSs). However, the timing and levels of protein released remain unclear. The present study aimed to characterize the secretion of the late pro-inflammatory cytokine HMGB1 by liver L02 and HepG2 cells. The human mononuclear macrophage cell line U937 was used as a control. Various concentrations of LPS were added to human U937, L02 and HepG2 cells for different durations, and the cells were analyzed at different time-points following this addition. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure cellular HMGB1 mRNA levels, western blotting was performed to detect HMGB1 in cellular supernatants and the translocation of HMGB1 from the nucleus to the cytosol was examined using immunofluorescence staining. L02 and HepG2 cells exhibited higher HMGB1 mRNA levels compared with the control U937 cells 20 and 24h following continuous exposure to LPS. U937cells exhibited higher HMGB1 mRNA levels compared with the corresponding L02 and HepG2 cells 16h following LPS exposure. The phase of HMGB1 protein detected in the cellular supernatants of L02 and HepG2 cells (16h) was later than that of U937 cells (8h). For the threecell lines, HMGB1 levels demonstrated a time dependency; however, the protein level was the highest in U937cells. In the threecell lines, translocation of HMGB1 from the nucleus to the cytosol occurred; however, the phases of HMGB1 translocation in L02 and HepG2 cells occurred later than in U937 cells. LPS-induced secretion of the late pro‑inflammatory cytokine HMGB1 by liver cells is characterized by a late phase of release and smaller quantity, and the process of HMGB1 secretion appears to be associated with HMGB1 translocation.

Ionic Complexation as a Non-Covalent Approach for the Design of Folate Anchored Rifampicin Gantrez Nanoparticles

The present study discloses the design of folate anchored Rifampicin-Poly methylvinylether maleic anhydride copolymer (Gantrez AN-119, Gantrez) nanoparticles (RFMGzFa) by ionic complexation. Folic acid was anchored to the preformed drug loaded nanoparticles. Folic acid was anchored in different concentration by simply varying the amount of folic acid added during preparation. RFMGzFa nanoparticles were prepared by emulsion solvent diffusion method. Gantrez AN-119 rapidly hydrolyzes in aqueous medium releasing carboxylic acid groups, to create an acidic environment. This facilitates protonation and subsequent ionic complexation of folic acid with the carboxylic groups, to enable anchoring. FTIR spectra confirmed this interaction. Infrared imaging revealed distribution of folic acid across the nanoparticle surface. Nanoparticles were obtained in the size range 350-450 nm with RFM loading of 12-14% w/w. Zeta potential confirmed colloidal stability. TEM/SEM revealed spherical morphology. RFMGzFa nanoparticles exhibited sustained release of RFM and folic acid. Folic acid showed sustained release upto 12 h, which was ion exchange mediated. A 480% enhancement in RFM uptake with RFMGzFa nanoparticles compared to 300% with RFMGz nanoparticles in-vitro, in human macrophage cell line U-937, suggested the role of folic acid in folate receptor mediated uptake. Ionic complexation represents a simple non-covalent approach for anchoring folic acid on polymeric nanoparticles of Gantrez.

S-Layer Protein Mediates the Stimulatory Effect of Lactobacillus helveticus MIMLh5 on Innate Immunity

The ability to positively affect host health through the modulation of the immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain. However, the identities of the bacterial cell components involved in cross talk with immune cells remain elusive. In this study, we characterized the dairy strain Lactobacillus helveticus MIMLh5 and its surface-layer protein (SlpA) using in vitro and ex vivo analyses. We found that MIMLh5 and SlpA exert anti-inflammatory effects by reducing the activation of NF-κB on the intestinal epithelial Caco-2 cell line. On the contrary, MIMLh5 and SlpA act as stimulators of the innate immune system by triggering the expression of proinflammatory factors tumor necrosis factor alpha and COX-2 in the human macrophage cell line U937 via recognition through Toll-like receptor 2. In the same experiments, SlpA protein did not affect the expression of the anti-inflammatory cytokine interleukin-10. A similar response was observed following stimulation of macrophages isolated from mouse bone marrow or the peritoneal cavity. These results suggest that SlpA plays a major role in mediating bacterial immune-stimulating activity, which could help to induce the host's defenses against and responses toward infections. This study supports the concept that the viability of bacterial cells is not always essential to exert immunomodulatory effects, thus permitting the development of safer therapies for the treatment of specific diseases according to a paraprobiotic intervention.

Acylated triterpenoid saponins from roots of Gypsophila trichotoma

Eleven new triterpenoid saponins were isolated from the roots of Gypsophila trichotoma Wend. (Caryophyllaceae) together with one known compounds. The structures were established on the basis of extensive 1D and 2D NMR analysis, completed by analysis of HR-ESI-MS and ESI-MSn. The cytotoxicity of the saponin extract from G. trichotoma was evaluated on a rat alveolar macrophage cell line NR8383 and a human macrophage cell line U937. The synergistic effect of the aminoacyl saponins, previously isolated from G. trichotoma, was tested for its ability to enhance the cytotoxicity of the targeted toxin in HER14 cells.

Identification of tripartite motif-containing 22 (TRIM22) as a novel NF-κB activator

Increasing evidence suggests that TRIM family proteins may play important roles in the regulation of innate immune signaling pathways. Here we report TRIM22 is involved in the activation of NF-κB. It was found that overexpression of TRIM22 could dose-dependently activate NF-κB as demonstrated by reporter gene assay and electrophoretic mobility shift assay, but had no effect on the activity of other transcription factors, including NF-AT, AP-1, C/EBP and IRFs. Further study showed that both the N-terminal RING domain and C-terminal SPRY domain were crucial for TRIM22-mediated NF-κB activation. Moreover, our results revealed that TRIM22 overexpression could significantly induce the secretion of pro-inflammatory cytokines by human macrophage cell line U937 in an NF-κB-dependent manner. These data suggested that TRIM22 was a positive regulator of NF-κB-mediated transcription.

Effect of colony morphology variation of Burkholderia pseudomallei on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro

BackgroundPrimary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. Variable morphology is associated with changes in the expression of a range of putative virulence factors. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Isogenic colony morphology types II and III were generated from 5 parental type I B. pseudomallei isolates using nutritional limitation. Survival of types II and III were compared with type I for all assays.ResultsMorphotype was associated with survival in the presence of H2O2 and antimicrobial peptide LL-37, but not with susceptibility to acid, acidified sodium nitrite, or resistance to lysozyme, lactoferrin, human neutrophil peptide-1 or human beta defensin-2. Incubation under anaerobic conditions was a strong driver for switching of type III to an alternative morphotype. Differences were noted in the survival and replication of the three types following uptake by human macrophages, but marked strain-to strain-variability was observed. Uptake of type III alone was associated with colony morphology switching.ConclusionsMorphotype is associated with phenotypes that alter the ability of B. pseudomallei to survive in adverse environmental conditions.

Induction of Proinflammatory Cytokines and C-Reactive Protein in Human Macrophage Cell Line U937 Exposed to Air Pollution Particulates

Exposure to particulate matter air pollution causes inflammatory responses and is associated with the progression of atherosclerosis and increased cardiovascular mortality. Macrophages play a key role in atherogenesis by releasing proinflammatory cytokines and forming foam cells in subendothelial lesions. The present study quantified the inflammatory response in a human macrophage cell line (U937) after exposure to an ambient particulate sample from urban dust (UDP) and a diesel exhaust particulate (DEP). The effect of native UDP and DEP was compared with their corresponding organic extracts (OE-UDP/OE-DEP) and stripped particles (sUDP/sDEP) to clarify their respective roles. Exposure to OE-UDP, OE-DEP, UDP, DEP, and 2,3,7,8-tetrachlorodibenzo-p-dioxin led to a greater increase of interleukin (IL)-8, tumor necrosis factor-α, and cyclooxygenase-2 mRNA expression than did the stripped particles, whereas sUDP, sDEP, UDP, and DEP led to a greater production of C-reactive protein and IL-6 mRNA. The particles and the organic extract-induced expression of cyclooxygenase-2 and cytochrome P450 (CYP)1a1 was significantly suppressed by co-treatment with an aryl hydrocarbon receptor (AhR) antagonist, indicating that these effects are mainly mediated by the organic components, which can activate the AhR and CYP1a1. In contrast, the induction of C-reactive protein and IL-6 seems to be a particle-related effect that is AhR independent. The inflammatory response induced by particulate matter was associated with a subsequent increase of cholesterol accumulation, a hallmark of foam cells. Together, these data illustrate the interaction between particulate matter and the inflammatory response as well as the formation of cholesterol-accumulating foam cells, which are early markers of cardiovascular disease.

P38 MAPK-dependent and YY1-mediated chemokine receptors CCR5 and CXCR4 up-regulation in U937 cell line infected by Mycobacterium tuberculosis or Actinobacillus actinomycetemcomitans

We have found previously that the chemokine receptors CCR5 and CXCR4, which are the coreceptors of HIV, are up-regulated in human macrophage cell line U937 infected by Mycobacterium tuberculosis (MTB). This suggests another possibility to explain the co-infection of MTB and HIV. In order to detect the up-regulation of CCR5 and CXCR4 as a unique phenomenon of MTB infection or a ubiquitous phenomenon of pathogenic bacteria, we investigated the expression changes of these two chemokine receptors in macrophages attacked by another bacterium Actinobacillus actinomycetemcomitans (AA) (from mRNA level and protein level). To reveal the molecular mechanism of these expression changes, p38 MAPK special inhibitor SB203580 was used and the expression of CCR5 and CXCR4 negative regulator YY1 transfactor was analyzed. Finally, we conclude that the up-regulation of CCR5 and CXCR4 can at least partially contribute to the down-regulation of transfactor YY1 which is p38 MAPK pathway-dependent and this up-regulation has little relationship with MTB and HIV co-infection.

Augmentation of macrophage phagocytosis by modified arabinoxylan rice bran (MGN-3/biobran).

MGN-3/Biobran, modified arabinoxylan rice bran, has been shown to be a potent biological response modifier (BRM) as manifested by stimulation of different arms of the immune system such as NK, T and B cells; however, its effect on macrophages has not yet been studied. The effects of MGN-3 on macrophage function was examined in vitro using 3 models: human macrophage cell line U937, murine macrophage cell line RAW264.7, and murine peritoneal macrophages (P-M phi). Treatment with MGN-3 resulted in an increase in the percentages of attachment and phagocytosis of yeast by macrophages. The effect depends on the type of macrophage and the dose of MGN-3 applied. Macrophages also demonstrated enhancement in their spreading ability, post treatment with MGN-3. Results also showed that MGN-3, in a dose dependent manner (1, 10,100 microg/ml), significantly induced high levels of production of cytokines: TNF-alpha; and IL-6. In addition, MGN-3 significantly increased nitric oxide (NO) production. This data demonstrates that MGN-3 is a potent inducer of phagocytic function by macrophage, and suggests that MGN-3 is a useful agent for fighting microbial infection.

Effect of trichothecenes on host resistance to bacterial infection

The effects of trichothecenes on specific and non-specific host resistance were examined. A relative low dose of deoxynivalenol (DON) reduced host resistance to salmonellosis but not the non-specific host resistance. DON disturbed the production of pro-inflammatory cytokines such as TNF-α after infection in vivo. Experiments using human macrophage cell line U-937 revealed that DON and other 8-ketotrichothecens disturbed the synthesis of pro-inflammatory cytokines. When cells were cultured with these trichothecens in combination with lipopolissacharide (LPS), the production of TNF-α, IL-8 and IL-6 was superinduced. These results suggested that DON and other 8-ketotrichothecens have the potential to superinduce pro-inflammatory cytokines production after infection in the presence of lipopolysaccharide.