Following Feline Sarcoma Related (FER) Kinase in Immune Cells during Klebsiella Pneumonia (PNA)

Author(s):
Vladislav Dolgachev; Thomas Lanigan; MV Suresh; Boya Zhang; Krishnan Raghavendran; David Machado-Aranda

Background:

The role of Feline Sarcoma Related (FER) tyrosine kinase in lung injury and PNA has not been extensively studied, but remains highly intriguing given the results of Genome Wide Association Studies (GWAS) showing its protective effects in Sepsis and Adult Respiratory Distress Syndrome (ARDS). Our previous research in mouse models of lung contusion and PNA found that electroporation-mediated (EP) lung gene delivery of FER plasmid located the majority of its expression on lung epithelial cells, while survival was associated to a robust mobilization of innate immune cells evident during bronchial alveolar lavage (BAL).

Hypothesis:

We ask if EP of dsRED-FER plasmid could label these effector cells directly and follow their function during PNA challenge.

Methods:

We electroporated dsRED-FER and pSG5-FER plasmid to lungs of C57Bl6 unchallenged mice. 24h or 72h post EP samples were harvested including BAL fluid and lung parenchyma. In parallel, we induced pneumonia 6 h prior to EP by providing 500 CFU of Klebsiella sp via pharyngeal drop. BAL cells were used for flow cytometry (FC) and also viewed after fixing on slides for cytology. Lung parenchyma was processed for mRNA purification and gene expression analysis as well as FC cell quantification. We used a cocktail of antibodies to label monocytes, macrophages and neutrophils (Ly6C, F480 and Gr-1) and determine their positivity to dsRED.

Results:

We found that dsRED plasmid induced a robust immune response predominantly labeling monocytes in unchallenged mice. Not surprisingly its expression was more predominant in neutrophils during PNA challenge. Flow cytometry findings were in agreement with histological tissue analysis. Control empty plasmids did not differentiate their response from naïve untreated controls. While numerically more cells were labeled at 24 h in a mixed population, the overall intensity of dsRED signal was higher at 72 h and enriched in monocytes. With unlabeled FER EP no dsRED signal was found. Quantitative analysis of human FER gene overexpression in mice lungs revealed 1323 ± 561  fold change over untreated control, peaking at 24h post EP(p < 0.05; n=5, ANOVA).

Conclusions:

New dsRED human FER plasmid EP represents powerful tool in quantitation of immune response induced by bacterial pneumonia challenge. Our results indicate EP-mediated gene transfer of FER can occur directly to innate cells and may have a dual phase response. This could constitute a novel strategy for their reprogramming in favor of eliminating bacteria and improving survival.