Dysfunction of Nrf-2 in CF Epithelia Leads to Excess Intracellular H2O2 and Inflammatory Cytokine Production

Abstract

Cystic fibrosis is characterized by recurring pulmonary exacerbations that lead to the deterioration of lung function and eventual lung failure. Excessive inflammatory responses by airway epithelia have been linked to the overproduction of the inflammatory cytokine IL-6 and IL-8. The mechanism by which this occurs is not fully understood, but normal IL-1β mediated activation of the production of these cytokines occurs via H2O2 dependent signaling. Therefore, we speculated that CFTR dysfunction causes alterations in the regulation of steady state H2O2. We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized. Increases in H2O2 heavily contributed to the excessive IL-6 and IL-8 production in CF epithelia. Proteomic analysis of three in vitro and two in vivo models revealed a decrease in antioxidant proteins that regulate H2O2 processing, by ≥2 fold in CF vs. matched normal controls. When cells are stimulated, differential expression in CF versus normal is enhanced; corresponding to an increase in H2O2 mediated production of IL-6 and IL-8. The cause of this redox imbalance is a decrease by ∼70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2. Inhibition of CFTR function in normal cells produced this phenotype, while N-acetyl cysteine, selenium, an activator of Nrf-2, and the overexpression of Nrf-2 all normalized H2O2 processing and decreased IL-6 and IL-8 to normal levels, in CF cells. We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8. Treatment with antioxidants can ameliorate exaggerated cytokine production without affecting normal responses.

Atypical Activation of the Unfolded Protein Response in Cystic Fibrosis Airway Cells Contributes to p38 MAPK-Mediated Innate Immune Responses

“…Three general mechanisms have evolved to restore ER homeostasis-increase in the protein folding capacity, attenuation of translation, and upregulation of the degradation machinery (ER-associated degradation, autophagy) (43). It is important to appreciate that in addition to misfolding of mutated CFTR protein, many factors are likely to have an impact on proteostasis to trigger ER stress in CF, including oxidative stress (11,16,17,(44)(45)(46), chronic infection (47), and decreased functionality of ER stress relief mechanisms, such as autophagy (21). Nevertheless, the literature contains conflicting results concerning the presence of ER stress in CF and the potential contribution of ER stress to CF inflammatory disease.…”

“…Recently, two important stress response mechanisms have been reported to be dysfunctional in CF. The antioxidant response mediated by the TF NRF2 has been shown to be defective in CF cells, leading to increased oxidative stress and inflammatory responses (17). Autophagy, a catabolic process by which cells degrade dysfunctional content and organelles during periods of stress, is known to be activated by ER stress (61,62); in turn, autophagy has been reported to decrease ER stress (63).…”

“…In CF cells, many factors are likely to have an impact on proteostasis to trigger ER stress, including misfolded CFTR protein (15), chronic infection, and oxidant-antioxidant imbalance (16,17). Nevertheless, the literature is not unanimous and data are conflicting on whether ER stress is induced in CF (18,19).…”

Antibiotic and Anti-Inflammatory Therapies for Cystic Fibrosis

“…Nrf2, a transcription factor active in respiratory epithelia and pivotal to mitigating the acute inflammatory response, is deficient in CF cells (Chen et al 2008;Nichols et al 2009). Small molecules like the synthetic triterpenoids that target proinflammatory signaling abnormalities in CF cells may be candidates for clinical trials.…”

Rescue of Dysfunctional Autophagy Attenuates Hyperinflammatory Responses from Cystic Fibrosis Cells

“…Autophagy is known to target misfolded DF508 CFTR from the ER to autophagosomes for degradation, preventing its accumulation (13). Activation of ER stress pathways by oxidative stress and misfolded CFTR accumulation correlates with inflammation in CF (14,15). Autophagy has recently been iden-tified as dysfunctional in CF (16)(17)(18), which might lead to heightened activation of ER stress pathways with inflammatory consequences (19); however, a direct link between autophagy and inflammation in CF has not yet been established.…”