Istopathology on lungs and spleens was performed as previously described [24,25].Macrophage remedy and infection in vitroBone marrow-derived macrophages (BMDM) were generated as described [26] and RAW264.7 murine macrophage cell line was a present from Prof. Gordon Brown, University of Aberdeen, UK. 5×105 cells had been cultured in the presence of indicated concentrations of simvastatin or PBS for 24 hours. Cells had been then infected with L. monocytogenes (MOI=10) or Listeria mutant for LLO (LLO) for 1 hour within the presence of simvastatin (50 ) ?mevalonate (one hundred ). At the indicated time points, bacterial development in cultured cells was determined as previously described [24].Cytokine and nitric oxide measurementsFollowing remedy with simvastatin and IFN- (one hundred Units/ml) overnight, macrophages were infected with L. monocytogenes and incubated at 37 for 1 hour. Cytokines for instance IL-12p40, TNF- and IL-6 in cell culture supernatants were measured by sandwich ELISA while nitric oxide (NO) was detected by Griess reagent assay [24].Formula of 1207294-92-5 Supplies and MethodsMice and bacteriaC57BL/6 mice have been maintained under specific-pathogen-free conditions within the biomedical animal facility on the Wellness Sciences Faculty, University of Cape Town. Mice were aged (8-12 weeks) and sex-matched for each experiment. L. monocytogenes (EGDe strain) were utilized for infection [24] and L. monocytogenes LLO mutant strain and GFP-expressing L. monocytogenes (BUG2377: EGDe-GFP-Cr) was a present from T. Chakraborty (Institute of Health-related Microbiology, University ofCholesterol content material in macrophages and serumAfter statin therapy, macrophages have been stained for cholesterol working with filipin dye as previously reported [27]. Alternatively, cholesterol content material was measured in soluble supernatant of total cell lysates applying an enzymatic cholesterol assay kit based on manufacturer’s directions (Bioassay system) [28] or in serum applying industrial kit (KAT).Cell viability and cytotoxicityAfter statin remedy, cellular viability and cytotoxicity was measured by reduction of yellow 3-(4,5-dimethythiazol-2-PLOS One | plosone.orgRole of Statins against Listeriosisyl)-2,5-diphenyl tetrazolium salt (MTT) (Sigma-Aldrich) by mitochondrial succinate dehydrogenase enzyme of living cells as previously described [29].ResultsHost defense against Listeria monocytogenes is enhanced by simvastatin remedy in miceWe investigated the effect of simvastatin treatment on bacterial burden throughout the acute phase of L. monocytogenes infection in mice. Intraperitoneal administration of ten and 20 mg/kg/day of simvastatin (Figure 1A) resulted inside a 100-fold reduction of bacterial burden inside the liver and spleen at day 3 post-infection (Figure 1B).Formula of 212651-52-0 This reduction in bacterial burden was accompanied by well-defined, smaller hepatic microabscesses in simvastatin-treated mice as revealed by liver histopathology and subsequent quantification of lesion sizes (Figure 1C).PMID:24605203 In addition, L. monocytogenes infection significantly elevated serum cholesterol (Figure 1D) but not triglycerides (Figure 1E) levels when when compared with non-infected mice. In addition, simvastatin therapy alone has no impact on the levels of serum cholesterol in mice (Figure 1F). We subsequent tested pravastatin, a hydrophilic statin. Mice were treated intraperitoneally with pravastatin at 2 and 10 mg/kg/day as shown in Figure 2A. Pravastatin treatment showed a trend towards decreased bacterial loads in the spleen (Figure 2B) and liver (Figure 2C), when compared.