Transactivation of Inducible Nitric Oxide Synthase Gene by Kruppel-like Factor 6 Regulates Apoptosis during Influenza A Virus Infection

Abstract

Influenza A virus (flu) is a respiratory tract pathogen causing high morbidity and mortality among the human population. NO is a cellular mediator involved in tissue damage through its apoptosis of target cells and resulting enhancement of local inflammation. Inducible NO synthase (iNOS) is involved in the production of NO following infection. Although NO is a key player in the development of exaggerated lung disease during flu infection, the underlying mechanism, including the role of NO in apoptosis during infection, has not been reported. Similarly, the mechanism of iNOS gene induction during flu infection is not well defined in terms of the host transactivator(s) required for iNOS gene expression. In the current study, we identified Kruppel-like factor 6 (KLF6) as a critical transcription factor essential for iNOS gene expression during flu infection. We also underscored the requirement for iNOS in inducing apoptosis during infection. KLF6 gene silencing in human lung epithelial cells resulted in the drastic loss of NO production, iNOS promoter-specific luciferase activity, and expression of iNOS mRNA following flu infection. Chromatin immunoprecipitation assay revealed a direct interaction of KLF6 with iNOS promoter during in vitro and in vivo flu infection of human lung cells and mouse respiratory tract, respectively. A significant reduction in flu-mediated apoptosis was noted in KLF6-silenced cells, cells treated with iNOS inhibitor, and primary murine macrophages derived from iNOS knockout mice. A similar reduction in apoptosis was noted in the lungs following intratracheal flu infection of iNOS knockout mice.

Nedd8 Regulates Inflammasome-Dependent Caspase-1 Activation

“…After 24 h, PMA-containing medium was replaced with fresh complete medium, and cells were maintained for 48 h before treatments. Bone marrow-derived macrophages (BMDMs) were obtained from femurs and tibias of wild-type C57BL/6 mice and were cultured for 6 to 8 days as described elsewhere (40,54,55). Cells were plated in 12-well cell culture plates containing RPMI 1640, 10% FBS, 100 IU/ml penicillin, 100 g/ml streptomycin, and 20 ng/ml granulocyte-macrophage colony-stimulating factor.…”

“…Influenza A virus [IAV; A/PR/8/34 (H1N1)] was grown in the allantoic cavities of 10-day-old embryonated eggs (40,54). IAV was purified by centrifugation (two times) on discontinuous sucrose gradients (40,54). 293 cells were maintained in DMEM supplemented with 10% FBS, 100 IU/ml penicillin, 100 g/ml streptomycin.…”

“…RSV was purified by centrifugation (two times) on discontinuous sucrose gradients as described previously (40,(50)(51)(52)(53). Influenza A virus [IAV; A/PR/8/34 (H1N1)] was grown in the allantoic cavities of 10-day-old embryonated eggs (40,54). IAV was purified by centrifugation (two times) on discontinuous sucrose gradients (40,54).…”

Temporal determination of lung NO system and COX-2 upregulation following ischemia–reperfusion injury

“…The abnormally increase in the apoptotic rates, that were detected the highest at 12 and 48 hours following IR coinsided with the highest level of expression of iNOS, suggesting that iNOS-derived NO could be involved in the destructive events occurring in the ischemic lung. Indeed, injury may be affected by changes in cellular iNOS, as has been previously reported by Mgbemena et al [34]. In their findings in the mouse lung during flu infection, the authors claimed that iNOS is essential in inducing apoptosis during infection.…”

Human respiratory syncytial virus non-structural protein NS1 modifies miR-24 expression via transforming growth factor-β

“…In A549 cells infected with RSV, KLF6 regulates the induction of both TGF-b (Mgbemena et al, 2011) and inducible nitric oxide synthase (Mgbemena et al, 2013), inhibiting the cells in G 0 /G 1 phase and preventing apoptosis during RSV infection (Mgbemena et al, 2012). miR-24 inhibition is also known to block the cell cycle in A549 cells (Cheng et al, 2005).…”