Akt activator SC79 stimulates antibacterial nitric oxide generation in human nasal epithelial cells in vitro

Abstract

BackgroundThe role of Akt in nasal immunity is unstudied. Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) expressed in epithelial ciliated cells. Nitric oxide (NO) production by ciliated cells can have antibacterial and antiviral effects. Increasing nasal NO may be a useful antipathogen strategy in chronic rhinosinusitis (CRS). We previously showed that small‐molecule Akt activator SC79 induces nasal cell NO production and suppresses IL‐8 via the transcription factor Nrf‐2. We hypothesized that SC79 NO production may additionally have antibacterial effects.MethodsNO production was measured using fluorescent dye DAF‐FM. We tested effects of SC79 during co‐culture of Pseudomonas aeruginosa with primary nasal epithelial cells, using CFU counting and live–dead staining to quantify bacterial killing. Pharmacology determined the mechanism of SC79‐induced NO production and tested dependence on Akt.ResultsSC79 induced dose‐dependent, Akt‐dependent NO production in nasal epithelial cells. The NO production required eNOS and Akt. The NO released into the airway surface liquid killed P. aeruginosa. No toxicity (LDH release) or inflammatory effects (IL8 transcription) were observed over 24 h.ConclusionsTogether, these data suggest multiple immune pathways are stimulated by SC79, with antipathogen effects. This in vitro pilot study suggests that a small‐molecule Akt activator may have clinical utility in CRS or respiratory other infection settings, warranting future in vivo studies.

Effects of Akt Activator SC79 on Human M0 Macrophage Phagocytosis and Cytokine Production

“…Several studies have pointed toward defects in CF macrophage function [38][39][40], making this cell a potential target for CF respiratory infections. While some have reported altered receptor-dependent Akt signaling in CF cells [41][42][43], we did not observe alterations to Akt signaling in CF nasal epithelial cells using a small-molecule Akt activator [37], suggesting that this pathway may be intact and druggable in CF macrophages. Macrophages are important players in early airway innate immunity [44,45], and enhancing macrophage function may help combat infections in CF.…”

“…Nonetheless, while SC79 is a useful tool for investigating the Akt pathway, we believe it still requires further in vitro and in vivo testing considering its potential to impact cell proliferation. Nonetheless, we believe that our data here and in previous studies [35,37] suggest that acute SC79 application could have both anti-bacterial and anti-inflammatory effects with possibly limited cellular toxicity, at least as shown in our in vitro primary cell models in the setting of short-term (24-48 h) exposure. Limited, targeted delivery of an Akt activator (e.g., as a topical nasal rinse or spray for nasal infections, or as an inhaler for lung infections) may also be a strategy to minimize any undesired effects of activating this pathway systemically.…”

“…We next tested if the activation of Akt by SC79 also resulted in NO production in M0 macrophages, as observed in airway epithelial cells [ 35 , 37 ]. We used DAF-FM, a dye that fluoresces after it covalently reacts with NO, and its reactive degradation products [ 18 ].…”

“…While e/nNOS are both activated by Ca 2+ /calmodulin binding, the AKT phosphorylation of human eNOS and nNOS can also activate NO production independently of Ca 2+ [ 31 , 32 , 33 , 34 ], including in nasal and lung epithelial cells [ 35 , 36 , 37 ]. We hypothesized that one way to enhance macrophage phagocytosis in, for example, CF patient macrophages, might be to target Akt directly to enhance innate killing.…”

“…We previously demonstrated that SC79 acutely increased Akt’s phosphorylation at one of its activating sites (S473) in multiple types of lung and nasal epithelial cells [ 35 ]. The resulting Akt activation and NO production from these epithelial cells was bactericidal against the common respiratory opportunistic pathogen, Pseudomonas aeruginosa [ 37 ]. We hypothesized that the activation of Akt-dependent NO production in human macrophages may enhance bacterial phagocytosis, like the NO activated downstream of T2Rs.…”