L. K. McCandless1, Y. S. Yin1, V. Dolgachev1, S. Panicker1, M. Suresh1, M. Hemmila1, K. Raghavendran1, D. Machado-Aranda1 1University Of Michigan,Division Of Acute Care Surgery/Department Of Surgery,Ann Arbor, MI, USA
Introduction: Alternative treatments against antibiotic-resistant bacteria are being strongly investigated. Potentially, transient manipulations of the genome could induce a more efficient immune response. A recent Genome-wide Association Study demonstrated correlation of FER gene expression with survival among pneumonia (PNA) patients. This was confirmed by electroporation-mediated delivery of FER gene in a lethal murine model of combined lung contusion (LC) and PNA. Neither study was conceived to determine the mechanisms for these favorable outcomes. We propose that FER improves survival by recruitment of activated immune cells primed to remove bacteria from the lung.
Methods: C57/BL6 mice were inoculated with 500 CFU of Klebsiella pneumoniae. At 1-hr they received plasmid DNA encoding human (pFER) gene via pharyngeal drop followed by 8 electroporation pulses (EP) inducing its expression in the lung. We recorded survival data for pFER-EP and control groups (PNA-only; PNA/EP-empty vector and PNA/EP-Na+/K+-ATPase gene). In parallel experiments, animals were sacrificed at specific time points (24, 48, 72 hr), bronchial alveolar lavage (BAL) fluid and lung tissues were harvested; cellular subpopulations counted by flow cytometry; specific genes and signaling pathways were assessed by TaqMan/Western Blot and finally cytokines by ELISA.
Results: After pFER-EP; 5-day survival was markedly improved compared to PNA-only control (80% vs 20%; p < 0.05). Early significant numbers of inflammatory monocytes were only detected in BAL fluid from pFER-EP animals exhibiting known antibacterial markers Toll Receptor- 2 and 4 (TLR2 and TLR4) respectively; being the later more predominant. Both BAL cells and lung tissues had higher protein expression of phosphorylated STAT3 (p-STAT3), a transcription factor critical in bacterial removal. The increased levels of p-STAT3 correlated with the decreased expression of its chaperone Heat-Shock Protein 90 (HSP90). However at 72 hr, HSP90 and other proteosome genes (NRLP2, NRF2) dramatically increased to counter-regulate p-STAT3, inducing monocyte apoptosis and avoiding damage to surrounding tissues. BAL TLR4+ monocytes were found to highly express Nitric oxide synthase-2 (Nos-2), Resistin-like molecule α1 (Fizz1), Tumor Necrosis Factor-α (TNFα) and Interferon-γ (IFNγ) all important against bacterial infection. Additionally, pFER-EP was able to rescue neutrophilic response in TLR4-/- mice, via increased production of strong chemoattractant KC and counterbalanced by decoy receptor for advance glycosylation (sRAGE), and independent from TNFα and IFNγ levels.
Conclusion: Lung gene delivery of the FER improved pneumonia survival by early STAT3 phosphorylation and suppression of HSP90, in turn enhancing antibacterial TLR4+ monocytes. Additionally, FER expression can modulate neutrophilic response via KC and sRAGE cytokines independently from TLR4, TNFα and IFNγ, thus constituting a novel therapeutic strategy.