Previously, we reported that electroporation-mediated (EP) delivery of the FER gene improved survival in a combined trauma-pneumonia model. to existing antibiotic classes have been deployed. However, recent clinical reports Dexamethasone inhibitor show bacteria are also developing resistance to these agents, such as in the case of Colistin-resistant and Carbapenem-resistant pneumonia. Our data shows active involvement of FER in the recruitment and activation of inflammatory monocytes and macrophages, with corresponding modifications of known signaling transduction pathways that enhance bacterial clearance and therefore improve survival. Additionally, we show the presence of FER in human lungs with gross inflammation. These findings indicate a novel mechanism of inflammatory modulation within the lung. FER gene up regulation represents a promising avenue of research and therapeutic potential towards the resolution of severe bacterial pneumonia, complementary to traditional antibiotic therapy. MATERIALS AND METHODS Mouse model of Bacterial Pneumonia (PNA) Female C57BL/6 wild-type mice, 18-20 g weight (Charles River Laboratories, Wilmington, MA, USA) were used for bacterial infection experiments. Mice were housed under specific pathogen-free conditions and were allowed a 1-week acclimation period to Rabbit Polyclonal to KITH_HHV1 their new surroundings prior to use. All experiments were performed in accordance with National Institutes of Health guidelines for care and use of animals. Approval for all experimental work was obtained from the University of Dexamethasone inhibitor Michigan Committee on Use and Care of Animals (UCUCA) Protocol PRO00006392 and the Institutional Biosafety Committee (IBC) Protocol IBCA00000315. To induce bacterial pneumonia, we administered in C57/Bl6 mice, was used. The administration of the bacterial suspension was performed via deep oral hypopharynx injection under isoflurane anesthesia. Animals were allowed to recover spontaneously and transported back to Biosafety Level-2 (BSL2) housing where animal health and survival data were recorded every 8 h. Recombinant plasmid DNA pneumonia, at a 6 h time line if insult was present. Animals were sacrificed at 24 h and harvested lungs were processed as in Dean (2012)17 and using the following antibodies Gr-1-PE,CD11c-APC-Cy7, Dexamethasone inhibitor F4/80-AF488, CD11b-PE-Cy7, CD206-APC, phospho-STAT1 and phospho-STAT6 (BioLegend and BD Biosciences, San Jose, CA, USA). Obtained Dexamethasone inhibitor data were plotted and analyzed using the FlowJo software (Tree Star, Inc., Ashland, OR, USA). Western blot analysis Whole-lung extracts and nuclear fractions were run on 12.5% SDS-polyacrylamide gels, with detailed protocol for sample processing as described in our previous publications(18, 19). Polyvinylidene difluoride membranes were probed sequentially with rabbit anti-STAT3 or anti-phospho-STAT3 (1:2000); followed by horseradish peroxidase-conjugated anti-rabbit IgG antibodies (1:3000) (AbCAM, Cambridge, MA, USA). We detected actin as a loading control using mouse anti-actin (1:2000) (AbCAM) followed by secondary as described above. Proteins were visualized by chemiluminiscense (Super Signal West Pico chemiluminescent substrate, Pierce, Rockford, IL, USA) using a Kodak photo imager. In our previous report(13) we found that the peak of transgene expression of FER by EP delivery was at 24 h weaning off by 72 h, thus we performed lung sample collection using these time points for our Western Blot analysis. Phagocytosis Assay Phagocytosis assays were performed as described elsewhere(18). Briefly, BAL alveolar macrophages (AM?) were isolated from BAL and plated at a concentration of 2105 cells/well and cultured overnight in Dulbeccos Modified Eagle Medium. Wells were aspirated and replaced with 50 L serum-free medium. AM? were then incubated with fluorescein isothiocyanate (FITC)-labeled, heat-killed transcripts were assessed using quantitative PCR analysis (TaqMan) with pre-developed primers and probe sets (Applied Biosystems, Carlsbad, CA, USA). Quantification of the genes Dexamethasone inhibitor of interest was normalized to GAPDH and expressed as fold increases over the naive control for each treatment at each time point. Mouse model of Methicillin-resistant (MRSA) Bacterial Pneumonia (PNA) Female A/J mice with reported susceptibility to MRSA, 18 to 20 g weight, (Charles River Laboratories, Wilmington, MA, USA) were used for MRSA bacterial infection experiment. A nonlethal concentration of 107 CFU of MRSA (ATCC catalog number 4012) was used to induce pneumonia. Procedures for inoculation and electroporation were performed in similar fashion as preceding experiments. After recovery, animals.