The triterpenoid CDDO limits inflammation in preclinical models of cystic fibrosis lung disease

Abstract

Excessive inflammation in cystic fibrosis (CF) lung disease is a contributor to progressive pulmonary decline. Effective and well-tolerated anti-inflammatory therapy may preserve lung function, thereby improving quality and length of life. In this paper, we assess the anti-inflammatory effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) in preclinical models of CF airway inflammation. In our experiments, mice carrying the R117H Cftr mutation have significantly reduced airway inflammatory responses to both LPS and flagellin when treated with CDDO before inflammatory challenge. Anti-inflammatory effects observed include reduced airway neutrophilia, reduced concentrations of proinflammatory cytokines and chemokines, and reduced weight loss. Our findings with the synthetic triterpenoids in multiple cell culture models of CF human airway epithelia agree with effects previously described in other disease models (e.g., neoplastic cells). These include the ability to reduce NF-kappaB activation while increasing nuclear factor erythroid-related factor 2 (Nrf2) activity. As these two signaling pathways appear to be pivotal in regulating the net inflammatory response in the CF airway, these compounds are a promising potential anti-inflammatory therapy for CF lung disease.

Figures

Fig. 1.

NF-κB activation in human airway epithelia. A: relative NF-κB activation in human airway epithelial cells lacking CFTR (16HBE14o−AS). B: relative NF-κB activation in human airway epithelial cells with functional CFTR (16HBE14o−S). C: IL-8 promoter activity in human airway epithelial cell with (S) and without (AS) CFTR. D: nuclear protein content of NF-κB phosphorylated p65 subunit in cystic fibrosis (CF)-like human airway epithelial cells (16HBE14o−AS). *P < 0.05; #P < 0.01. Performed in 3 separate experiments, n = 2–4 replicates per experiment. Stim, stimulation; CDDO, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid; VEH, vehicle.

Fig. 2.

Nuclear factor erythroid-related factor 2 (Nrf2) activation in human airway epithelia. A: increased Nrf2 transcriptional activation above baseline (vehicle treatment) achieved by 300 nM CDDO exposure in human airway epithelial cells with (S) and without (AS) functional CFTR. Intracellular H2O2 concentration in human airway epithelia cells at air-liquid interface (ALI) with chemically inhibited CFTR (B) or functional CFTR (C). D: NAD(P)H:quinone oxidoreductase 1 (NQO1) activity in human airway epithelial cells homozygous for the ΔF508 CFTR mutation (CFBE41o−). *P < 0.05; #P < 0.01. Nrf2 reporter assays performed with 2 separate experiments, n = 6 replicates per experiment. H2O2 assay, n = 6 per condition. NQO1 assay, n = 4 per condition. I-172, CFTRinh-172.

Fig. 3.

IL-8 secretion in primary human airway epithelia IL-8 secretion from primary human airway epithelial cells at ALI treated with chemical CFTR inhibitor, with and without exposure to 300 nM CDDO before stimulation with TNFα. *P < 0.05. Performed in 3 separate experiments, total n = 7–9 replicates per condition.

Fig. 4.

Mean daily weight of CF mice treated with CDDO or vehicle control before and after LPS challenge.

Fig. 5.

Bronchoalveolar lavage (BAL) neutrophilia and proinflammatory mediators. A: neutrophil percentage of leukocytes in response to intratracheal LPS or flagellin in CF mice. *P < 0.001. Fold difference in the concentration of cytokines and chemokines in BAL of CF mice (B) and wild-type (WT) mice (C) in response to LPS stimulation are shown. *P < 0.001; †P < 0.01; #P < 0.05. PMN, polymorphonuclear neutrophils; FLG, flagellin; MIP-2, macrophage inflammatory protein-2; KC, keratinocyte-derived chemokine.