Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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NF-?B Activation Controls Phagolysosome Fusion-Mediated killing of Mycobacteria by Macrophages

ABSTRACT: Mycobacterium smegmatis is a model non-pathogenic mycobacterium that is efficiently killed by macrophages. Here, we explore the role of NF-?B in the innate immune response, focusing in detail on the mechanisms of the first killing period (1-4h) of M. smegmatis which coincides with phagosome-lysosome fusion. We show that infection of macrophages with M. smegmatis induces an activation of NF-?B and this activation is required for killing since treatment of macrophages with NF-?B inhibitors or siRNA silencing of the NF-?B subunit p65 increases bacterial survival. NF-?B induced proteins were thus hypothesized to be essential during the first phase of M. smegmatis killing. We therefore identified, using RNA microarray, the genes that were regulated during infection in the absence and presence of NF-?B inhibitors. By subtraction this provided a list of pro-inflammatory proteins that were under the control of NF-?B and putatively involved in the killing response. Among these category of genes were those for lysosomal enzymes and membrane trafficking regulators, including Cathepsins, LAMP-2 and Rab34, are regulated by NF-?B. Moreover, inhibition of NF-?B signaling retarded the delivery of v-ATPase, LAMP-2, CtsZ and CtsH thereby impairing the maturation of mycobacterial phagosomes. Collectively; our data provide the first compelling evidence that the innate immune response via NF-?B activation is linked to phagosome fusion with lysosomes that is essential for killing of mycobacteria. Keywords: NFkB inhibitor treated vs untreated The study includes J774 macrophage cell lines which are infected with Mycobacterium smegmatis in the presence and absense of NFkB inhibitor SC-514.Total RNA was isolated from individual samples and each sample was hybridised to CodeLink Mouse whole genome bioarray slides.This study was intended to know the role of NFkB regulated genes in killing non-pathogenic mycobacteria during 4 hour post infection of macrophages.

ORGANISM(S): Mus musculus

SUBMITTER: Bibhuti Mishra

PROVIDER: E-GEOD-8999 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The receptors engaged during phagocytic particle uptake determine the signaling events that occur during phagosome formation and maturation. However, pathogens generally have multiple ligands, making it difficult to dissect the roles of individual receptors in these processes. Here, we used latex beads coupled to single ligands, focusing on IgG, mannan, LPS and avidin, and monitored phagocytic uptake rates, phago-lysosomal fusion events, macrophage gene expression and the proteomic composition of isolated phagosomes. The pattern of gene expression and the protein composition of isolated phagosomes showed that each bead ligand altered a distinct pattern of genes and led to a different composition of phagosomes. These data argue that activation of each receptor initiates a specific signature of signaling events that last many hours and influences several phagocytosis functions. All samples and controls were carried out in triplicates. J774.A1 cells (mouse macrophage-like cell line) were seeded onto 6-well plates one day before the experiment. Subsequently, cells were incubated with serum-free DME medium containing 0.01 % latex beads of 1 Âµm diameter coupled to the following ligands: avidin (Av), the Fc fragment of mouse IgG (Fc), lipopolysaccharides from Klebsiella pneumoniae (LPS) and mannan from Saccharomyces cerevisae (Man) for 30 and 60 minutes. After incubation, samples as well as untreated controls were washed twice in PBS and total RNA was extracted using RNeasy kit (Qiagen) following manufacturer's instructions. Each sample was hybridized to CodeLink Mouse whole genome bioarray slides (Amersham). This study was intended to analyze the role of receptor-ligand interactions on phagosome maturation and gene expression after receptor-mediated phagocytosis in macrophages. CodeLink EXP v4.0-processed data are represented in the Sample tables, GeneSpring-processed data are linked as a supplementary files to the matrix table, additional results available as a supplementary file on the Series record. http://www.biologie.uni-rostock.de/tierphysiologie/agkuznetsov.html

Project description:Mouse macrophages J774A.1 were pre-treated with the anti-inflammatory lipid eicosapenaenoic acid (EPA) or the pro-inflammatory lipid ceramide (Cer) for 3h. Macrophages were then infected with Mycobacterium smegmatis for 1h, and total RNA was collected. In a parallel experiment, infected macrophages were infected for 1h, 4h and 24h. At these time points, macrophages were lysed, bacteria was collected and quantified by the Colony Forming Units (CFU) Assay. This provided the kinetics of the killing of Mycobacteria inside mouse macrophages. CFU experiments revealed that cells pre-treated with EPA showed an increased number of bacteria inside macrophages, in contrast to cells pre-treated with Cer. To dissect the molecular mechanisms involved in the survival and killing of mycobacteria infected macrophages, mediated by lipids, gene expression studies were performed. Cultures of Mycobacterium smegmatis mc2155 harbouring a p19-(long lived) EGFP plasmid were grown to exponential growth phase. Bacteria were pelleted, washed in PBS and resuspended in medium DMEM with a multiplicity of infection (MOI) of 10 (10 bacteria per macrophage). Clumps of bacteria were removed by ultrasonic treatment of bacterial suspensions in an ultrasonic water bath for 15 minutes, followed by a low speed centrifugation for 2 minutes. Mouse macrophages J774A.1 were pre-.treated with the anti-inflammatory lipid eicosapenaenoic acid (EPA) or the pro-inflammatory lipid ceramide (Cer), 3h before infection. Mouse macrophages J774A.1 were infected with Mycobacterium smegmatis mc2155 for 1h, at 37M-BM-:C and 5% CO2. After 1h of infection, cells were washed with PBS. After washing, 1 ml Trizol was added per well, to collect total RNA. The experimental condition were: samples 1.1, 1.2, 1.3: Untreated macrophages; samples 2.1, 2.2, 2.3: Mock treated macrophages (ethanol 1ul/ml); samples 3.1, 3.2, 3.3: EPA (15uM) treated macrophages (ethanol 1ul/ml); samples 4.1, 4.2, 4.3: Cer (5ug/ml) treated macrophages (ethanol 1ul/ml); samples 5.1, 5.2, 5.3: Untreated macrophages, infected with Mycobacterium smegmatis mc2155 for 1h; samples 6.1, 6.2, 6.3: Mock treated macrophages (ethanol 1ul/ml) infected with Mycobacterium smegmatis mc2155 for 1h; samples 7.1, 7.2, 7.3: EPA (15uM) treated macrophages (ethanol 1ul/ml) infected with Mycobacterium smegmatis mc2155 for 1h; samples 8.1, 8.2, 8.3: Cer (5ug/ml) treated macrophages (ethanol 1ul/ml) infected with Mycobacterium smegmatis mc2155 for 1h; In a parallel experiment, infected macrophages remained with the bacteria for 1h, 4h and 24h after infection. After these time points, the macrophages were lysed, the bacteria was collected and quantified by the Colony Forming Units (CFU) Assay. This provided the kinetics of the killing of non-pathogenic intracellular bacteria inside mouse macrophages. For the CFU assay, after 1h of infection, cells were washed with PBS and Gentamicin (10ug/ml) in DMEM to kill the extracellular bacteria. At discrete time points, cells were washed with PBS and lysed with sterilized water. Quantitative cultures of bacteria were performed in a 10-fold serial dilutions, inoculated on 7H10 agar plates. 5ul were plated in triplicate and the number of colonies were counted after 48h.

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NF-?B Activation Controls Phagolysosome Fusion-Mediated killing of Mycobacteria by Macrophages

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