NRF2 and NLRP3 gene activation rescues inflammatory response in TLR-4 deficient animals after FER electroporation

Author(s):
Vladislav Dolgachev; Boya Zhang; Sanjay Balijepalli; Samantha Swamy; Sreehari Panicker; MV Suresh; Krishnan Raghavendran; David Machado-Aranda

Background:

Introduction: Toll-like receptors are important components of immune responses to microbial products. TLR4 deficiency results in tremendously accelerated mortality after pneumonia challenge. We recently described that airway electroporation of human FER gene could rescue mice upon lethal challenge combining lung contusion and pneumonia. Here we set to study whether acute overexpression of FER in lung tissue could rescue TLR4 deficient mice (24h 100% mortality upon pneumonia) exposed to bacterial pneumonia

Hypothesis:

Hypothesis: We recently described that overexpression of FER gene uses a Signal Transduction and Transciption-3 (STAT-3) pathway to improve survival in a pneumonia model. FER will activate downstream STAT-3 signal transduction mechanisms despite TLR-4 deficiency and rescue the lung inflammatory response

Methods:

Methods: TLR-4 ^-/- mice were subject to an initial unilateral lung contusion injury and later a 6 h were inoculated with 500 CFU of Klebsiella sp. In between insults they received a DNA plasmid encoding human FER (PNA/pFER-EP) via pharyngeal drop followed by 8 electroporation pulses (EP) inducing FER expression in the lung. Bronchial alveolar lavage (BAL) and lung samples were processed while cellular subpopulations, bacterial CFUs, histology, gene and protein expression, and cytokines were analyzed.

Results:

Results: Acute overexpression of FER gene in lung tissue significantly extended survival of TLR4^-/- mice upon bacterial pneumonia challenge (0%/48h vs 40%/96h). In contrast to wild type animals, activated STAT-3 and TNF-α levels were surprisingly depressed despite FER electroporation treatment. However, transcription factor NRF2 and inflammasome component NLRP3 were significantly overexpressed, allowing the maturation and secretion of IFN-γ, KC and CXCL2 and thus enhanced leukocyte recruitment. TLR4^-/- were able to survive 48 h longer, time until transient gene expression of FER disappears.

Conclusions:

Conclusion: Manipulation of immune response with short-lived FER overexpression in resolution of lung contusion complicated with bacterial pneumonia rescued deficient in TLR4^-/- mice monocytes and neutrophils recruitment into an airway. FER overexpression significantly extended survival of TLR4^-/- mice upon lethal pneumonia challenge. FER drives prolonged expression of Nrf2 gene in BAL cells of pneumonia challenged mice.