Classical and alternative nuclear factor-kappa B in lung epithelium: Impacts in allergic airway disease and avenues for redox regulation

Nuclear Factor kappaB (NF-kappaB) is a transcription factor whose activation is increased in settings of allergic asthma. At least two parallel NF-kappaB pathways exist: the classical pathway, which plays a role in inflammation and cell survival, and the alternative pathway, which regulates lymphoid cell development and organogenesis. The classical NF-kappaB pathway regulates inflammatory responses derived from lung epithelial cells; however, the role of the alternative pathway in lung epithelial cells remains unclear. We demonstrate that both classical and alternative NF-kappaB are activated in lung epithelial cells in response to multiple pro-inflammatory agonists, and siRNA-mediated knockdown of alternative NF-kappaB proteins largely attenuates pro-inflammatory cytokine production. Furthermore, simultaneous activation of both pathways leads to cooperative increases in pro-inflammatory responses, indicating a potential role for both classical and alternative NF-kappaB in the regulation of epithelial-derived pro-inflammatory responses.;NF-kappaB activation in the epithelium modulates allergic inflammation in mouse models of allergic airway disease, however, its role in the context of an allergen relevant to human asthma remains unknown. In order to address the impact of inhibition of NF-kappaB in the epithelium in vivo, we utilized a House Dust Mite (HDM)-induced model of allergic airway disease. We demonstrate that HDM exposure activates classical and alternative NF-kappaB in both murine lung epithelium and human bronchial epithelial cells. Furthermore, following exposure to HDM, airway hyperresponsiveness, neutrophilic inflammation, and remodeling are attenuated in transgenic CC10-NF-kappaB SR (airway epithelial specific inhibitor of classical and alternative NF-kappaB) mice in comparison to wild type mice. Our data also demonstrate that specific knockdown of the alternative NF-kappaB protein, RelB, in the lung partially protects against HDM-induced pro-inflammatory responses, indicating that both classical and alternative NF-kappaB are important in HDM-induced responses.;NF-kappaB proteins are modified by the redox-dependent post-translational modification, S-glutathionylation, under conditions of oxidative stress. S-glutathionylation of IKKbeta, an upstream kinase in the NF-kappaB pathway, is known to decrease its catalytic activity; however, it is unknown how S-glutathionylation of IKKbeta occurs. GSTP is an enzyme that catalyzes protein S-glutathionylation under conditions of oxidative stress and has been associated with the development of allergic asthma. We aimed to determine whether GSTP regulates NF-kappaB signaling, S-glutathionylation of IKKbeta, and pro-inflammatory cytokine production. We demonstrate that siRNA-mediated knockdown of GSTP modulates NF-kappaB activation, NF-kappaB transcriptional activity, and pro-inflammatory cytokine production in response to LPS, a component of a bacterial cell wall. Furthermore, we demonstrate that GSTP associates with IKKbeta in response to agonist stimulation and dampens IKKbeta-induced pro-inflammatory cytokine production, surprisingly, independent of its catalytic activity. We also show that GSTP associates with other proteins of the NF-kappaB pathway, indicating a potential dual mechanism for repression of NF-kappaB-induced signaling. These studies collectively demonstrate that classical and alternative NF-kappaB contribute to epithelial-derived inflammatory responses, and GSTP may be a novel target by which NF-kappaB can be regulated.