UPR modulation of host immunity by Pseudomonas aeruginosa in cystic fibrosis

Cystic fibrosis (CF) is a progressive multi organ autosomal recessive disease with devastating impact on the lungs caused by derangements of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Morbidity and mortality in CF are largely caused by lung complications due to the triad of impaired mucociliary clearance, microbial infections and chronic inflammation. P. aeruginosa is a main respiratory pathogen in individuals with CF infecting most patients in later stages. Despite its recognized clinical impact, the molecular mechanisms that underlie P. aeruginosa pathogenesis and the host response to P. aeruginosa infection remain incompletely understood. The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR γ ) an important modulator of inflammation and host defense, has shown to be reduced in CF airways. In this study we sought to investigate the upstream mechanisms repressing PPAR γ expression and its impact on airway epithelial host defense. Endoplasmic Reticulum-stress (ER-stress) triggered unfolded protein response (UPR) activated by misfolded CFTR and P. aeruginosa infection contributed to attenuated expression of PPAR γ . Specifically, the PERK signaling pathway lead to the enhanced expression of the CCAAT-enhancer-binding-protein homologous protein (CHOP). CHOP induction led to the repression of PPAR γ expression. Mechanistically, we show that CHOP induction mediated PPAR γ attenuation, impacted the innate immune function of normal, ΔF508 primary airway epithelial as well as bronchial epithelial cells by reducing expression of anti-microbial peptide (AMP) and paraoxanse-2 (PON-2), as well as enhancing IL-8 expression. Furthermore, mitochondrial reactive oxygen species production (mt-ROS) and ER stress positive feedforward loop also dysregulated mitochondrial bioenergetics. Additionally, our treating cells with PPAR γ agonists attenuated mitochondrial dysfunction and enhanced host defenses to P. aeruginosa. These data are the first to delineate a potential mechanism by UPR induced attenuation of PPAR γ alters host defense by P. aeruginosa CF epithelial cells. THP-1 derived macrophages, enhanced barrier function macrophage post-infection the The results of our findings indicate that UPR induction by non-mucoid and mucoid CF variants of P. aeruginosa inhibit host defense by attenuating PPAR γ expression, mitochondrial bioenergetics and enhancing mt-ROS. Specifically, induction of the UPR, PERK signaling arm by P. aeruginosa CF variants, enhances CHOP expression causing transcriptional repression of PPAR γ . This axis increases inflammation through induction of IL-8 expression and suppresses host defense via downregulation of the AMP HBD-1, and the quorum quenching enzyme PON-2. Moreover, induction of ER-stress by P. aeruginosa also enhances mt-ROS and CHOP mediated PPAR γ downregulation potentially contributes to mitochondrial dysfunction, since PPAR γ agonist PIO partially restores mitochondrial function. Normal and CF primary airway epithelial as well as BEAS-2B cells pre-treated with PIO significantly reduces IL-8 and enhances HBD-1 and PON-2 expression. Our findings are the first to study the impact of infection induced ER stress on host defense and mitochondrial dysfunction in CF. and mitochondrial protein (91, 92), (iv) dysregulation of mitochondrial bioenergetics. Our data are the first to show how UPR induced cellular stress modulates epithelial host response by attenuating PPAR γ and provide novel insights into the mechanisms by which PPAR γ agonists can enhance host immune function in CF disease exacerbation. PPAR γ agonists are currently in clinical trials for management of inflammation in CF patients Inhibition of ER stress 95) as well as, restoration of mitochondrial glutathione depleted by ER stress could potentially provide novel approaches to enhance immune and metabolic benefits to CF disease management. basal protein levels were not altered (H and I) as compared to the mRNA levels. Infection with P. aeruginosa significantly reduced PON-2 expression in ΔF508 cells. Significant reduction of PON-2 mRNA (J) and protein expression (K and L) was also noted in BEAS-2B cells infected with the P. aeruginosa strains. Results are mean SEM. *p<0.05, **P< and or 5 independent experiments, t-test was performed transfected and tunicamycin treated cells also showed a decrease, (C) . Quantitation of PPAR γ mRNA expression in Si CHOP, revealed a significant increase (B) , whereas tunicamycin and Ad CHOP (B) significantly attenuated PPAR γ mRNA. Protein expression of PPAR γ was concurrently, significantly increased and decreased with Si CHOP and tunicamycin (C and D) , whereas CHOP protein expression was significantly decreased and increased respectively, (C and D). Impact of CHOP mediated decrease in PPAR γ was evaluated by enumerating the mRNA HBD-1 (E) , which was significantly enhanced by Si CHOP (E) , corresponding to increased PPAR γ expression. Results are mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001; #P<0.05, ###P<0.001; $$$P<0.001 and &&&P<0.001; n=3 independent experiments. and expression levels were similarly decreased with aeruginosa but enhanced with PIO pre-treatment (C and D) . In parallel with mRNA, protein levels of PPAR γ (E and F) and PON-2 (E and G) were significantly restored in a similar manner (E) . Samples obtained from Normal, ΔF508 primary bronchial epithelial and BEAS-2B respectively were further analyzed for the impact of PIO on the innate immune function. PIO restored the expression of HBD-1 in the non-CF, ΔF508 primary epithelia (H) as well as BEAS-2B cells, respectively (J) . Furthermore, pre-treatment of primary epithelia (I) and BEAS-2B cells (K) , respectively, significantly attenuated the production of IL-8 post-treatment with P. aeruginosa. Results are mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001, #P<0.05, ##P<0.01 and ###P<0.001; n=3 independent experiments.