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Azithromycin selectively reduces tumor necrosis factor alpha levels in cystic fibrosis airway epithelial cells.

ABSTRACT: Azithromycin (AZM) ameliorates lung function in cystic fibrosis (CF) patients. This macrolide has been suggested to have anti-inflammatory properties as well as other effects potentially relevant for therapy of CF. In this study, we utilized three CF (IB3-1, 16HBE14o- AS3, and 2CFSMEo-) and two isogenic non-CF (C38 and 16HBE14o- S1) airway epithelial cell lines to investigate whether AZM could reduce tumor necrosis factor alpha (TNF-alpha) mRNA and protein levels by real-time quantitative PCR analysis and an enzyme-linked immunosorbent assay (ELISA), respectively. We studied the effects on the DNA binding of NF-kappaB and specificity protein 1 (Sp1) by an ELISA. Non-CF cells express significantly lower TNF-alpha mRNA and protein levels than an isogenic CF cell line. In CF cells, AZM treatment causes a 30% reduction of TNF-alpha mRNA levels (P < 0.05) and a 45% decrease in TNF-alpha secretion (P < 0.05), reaching approximately the levels of the untreated isogenic non-CF cells. In CF cells, NF-kappaB and Sp1 DNA binding activities were also significantly decreased (about 45 and 60%, respectively; P < 0.05) after AZM treatment. Josamycin, a macrolide lacking clinically described anti-inflammatory effects, was ineffective. Finally, AZM did not alter the mRNA expression levels of interleukin-6, a proinflammatory molecule not differentially expressed in CF and isogenic non-CF cells. The results of our study support the anti-inflammatory activities of this macrolide, since we show that AZM reduced the levels of TNF-alpha and propose inhibitions of NF-kappaB and Sp1 DNA binding as possible mechanisms of this effect.

SUBMITTER: Cigana C

PROVIDER: S-EPMC1803122 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Azithromycin selectively reduces tumor necrosis factor alpha levels in cystic fibrosis airway epithelial cells.

Cigana Cristina C Assael Baroukh Maurice BM Melotti Paola P

Antimicrobial agents and chemotherapy 20070108 3

Azithromycin (AZM) ameliorates lung function in cystic fibrosis (CF) patients. This macrolide has been suggested to have anti-inflammatory properties as well as other effects potentially relevant for therapy of CF. In this study, we utilized three CF (IB3-1, 16HBE14o- AS3, and 2CFSMEo-) and two isogenic non-CF (C38 and 16HBE14o- S1) airway epithelial cell lines to investigate whether AZM could reduce tumor necrosis factor alpha (TNF-alpha) mRNA and protein levels by real-time quantitative PCR an ...[more]

PMID: 17210769

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Project description:BACKGROUND:Defective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF). Abnormal airway iron homeostasis and the presence of lipid peroxidation products, indicative of oxidative stress, are features of CF lung disease. RESULTS:Here, we report that CF AECs (IB3-1) are susceptible to ferroptosis, a type of cell death associated with iron accumulation and lipid peroxidation. Compared to isogenic CFTR corrected cells (C38), the IB3-1 cells showed increased susceptibility to cell death upon exposure to iron in the form of ferric ammonium citrate (FAC) and the ferroptosis inducer, erastin. This phenotype was accompanied by accumulation of intracellular ferrous iron and lipid peroxides and the extracellular release of malondialdehyde, all indicative of redox stress, and increased levels of lactate dehydrogenase in the culture supernatant, indicating enhanced cell injury. The ferric iron chelator deferoxamine (DFO) and the lipophilic antioxidant ferrostatin-1 inhibited FAC and erastin induced ferroptosis in IB3-1 cells. Glutathione peroxidase 4 (GPX4) expression was decreased in IB3-1 cells treated with FAC and erastin, but was unchanged in C38 AECs. Necroptosis appeared to be involved in the enhanced susceptibility of IB3-1 AECs to ferroptosis, as evidenced by partial cell death rescue with necroptosis inhibitors and enhanced mixed lineage kinase domain-like (MLKL) localisation to the plasma membrane. CONCLUSION:These studies suggest that the increased susceptibility of CF AECs to ferroptosis is linked to abnormal intracellular ferrous iron accumulation and reduced antioxidant defences. In addition, the process of ferroptotic cell death in CF AECs does not appear to be a single entity and for the first time we describe necroptosis as a potential contributory factor. Iron chelation and antioxidant treatments may be promising therapeutic interventions in cystic fibrosis.

Project description:Introduction: The responses of cystic fibrosis (CF) airway epithelial cells (AEC) to rhinovirus (RV) infection are likely to contribute to early pathobiology of lung disease with increased neutrophilic inflammation and lower apoptosis reported. Necrosis of AEC resulting in airway inflammation driven by IL-1 signaling is a characteristic finding in CF detectable in airways of young children. Being the most common early-life infection, RV-induced epithelial necrosis may contribute to early neutrophilic inflammation in CF via IL-1 signaling. As little is known about IL-1 and biology of CF lung disease, this study assessed cellular and pro-inflammatory responses of CF and non-CF AEC following RV infection, with the hypothesis that RV infection drives epithelial necrosis and IL-1 driven inflammation. Methods:Primary AEC obtained from children with (n = 6) and without CF (n = 6) were infected with RV (MOI 3) for 24 h and viable, necrotic and apoptotic events quantified via flow cytometry using a seven-step gating strategy (% total events). IL-1?, IL-1?, IL-1Ra, IL-8, CXCL10, CCL5, IFN-?, IL-28A, IL-28B, and IL-29 were also measured in cell culture supernatants (pg/mL). Results:RV infection reduced viable events in non-CF AEC (p < 0.05), increased necrotic events in non-CF and CF AEC (p < 0.05) and increased apoptotic events in non-CF AEC (p < 0.05). Infection induced IL-1? and IL-1? production in both phenotypes (p < 0.05) but only correlated with necrosis (IL-1?: r = 0.80; IL-1?: r = 0.77; p < 0.0001) in CF AEC. RV infection also increased IL-1Ra in non-CF and CF AEC (p < 0.05), although significantly more in non-CF AEC (p < 0.05). Finally, infection stimulated IL-8 production in non-CF and CF AEC (p < 0.05) and correlated with IL-1? (r = 0.63 & r = 0.74 respectively; p < 0.0001). Conclusions:This study found RV infection drives necrotic cell death in CF AEC. Furthermore, RV induced IL-1 strongly correlated with necrotic cell death in these cells. As IL-1R signaling drives airway neutrophilia and mucin production, these observations suggest RV infection early in life may exacerbate inflammation and mucin accumulation driving early CF lung disease. Since IL-1R can be targeted therapeutically with IL-1Ra, these data suggest a new anti-inflammatory therapeutic approach targeting downstream effects of IL-1R signaling to mitigate viral-induced, muco-inflammatory triggers of early lung disease.

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Azithromycin selectively reduces tumor necrosis factor alpha levels in cystic fibrosis airway epithelial cells.

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Azithromycin selectively reduces tumor necrosis factor alpha levels in cystic fibrosis airway epithelial cells.

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