ABSTRACT: 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.
