Dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) leads to many cellular consequences, including perinuclear accumulation of free cholesterol due to impaired endosomal transport. The hypothesis being tested is that CF-related perinuclear cholesterol accumulation due to disrupted endocytic trafficking occurs as a result of reduced microtubule (MT) acetylation. Here, it is identified that acetylated-α-tubulin (Ac-tub) content is reduced by ∼40% compared with respective wild-type controls in both cultured CF cell models (IB3) and primary Cftr−/− mouse nasal epithelial tissue. Histone deacetylase 6 (HDAC6) has been shown to regulate MT acetylation, which provides reasonable grounds to test its impact on reduced Ac-tub content on CF cellular phenotypes. Inhibition of HDAC6, either through tubastatin treatment or HDAC6 knockdown in CF cells, increases Ac-tub content and results in redistributed free cholesterol and reduced stimulation of NF-κB activity. Mechanistically, endoplasmic reticulum stress, which is widely reported in CF and leads to aggresome formation, is identified as a regulator of MT acetylation. F508del CFTR correction with C18 in primary airway epithelial cells restores MT acetylation and cholesterol transport. A significant role for phosphatidyl inositol-3 kinase p110α is also identified as a regulator of MT acetylation.

The goal of this study was to identify a mechanism regulating cholesterol accumulation in cystic fibrosis (CF) cells. Both CFTR activation and expression are regulated by the cAMP pathway, and it is hypothesized that a feedback response involving this pathway may be involved in the phenotype of cholesterol accumulation. To examine the role of the cAMP pathway in cholesterol accumulation, we treated two CF model cell lines with the Rp diastereomer of adenosine 3′,5′-cyclic monophosphorothioate ( Rp-cAMPS) and visualized by filipin staining. Rp-cAMPS treatment eliminated cholesterol accumulation in CF cells, whereas 8-bromo-cAMP treatment led to cholesterol accumulation in wild-type cells. To confirm these findings in an independent model system, we also examined the role of cAMP in modulating cholesterol accumulation in Niemann-Pick type C (NPC) fibroblasts. Expression of the protein related to NPC, NPC1, is also directly regulated by cAMP; therefore, it is postulated that NPC cells exhibit the same cAMP-mediated control of cholesterol accumulation. Cholesterol accumulation in NPC cells also was reduced by the presence of Rp-cAMPS. Expression of β-arrestin-2 (βarr2), a marker of cellular response to cAMP signaling, was significantly elevated in CF model cells, Cftr^−/−MNE, primary tissue obtained by nasal scrapes from CF subjects, and in NPC fibroblasts compared with respective controls.

Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.

The hypothesis of this study was that Hdac6 depletion would restore cystic fibrosis (CF) responses to bacterial challenge to more wild type profiles using a CF mouse model. CF mice harboring the F508del Cftr mutation respond to bacterial challenge with 25,000 CFU Pseudomonas aeruginosa embedded into agarose beads to slow clearance. CF mice respond significantly more aggressively to this challenge compared to WT mice with respect to bacterial clearance, weight loss, neutrophil recruitment, and MIP-2 production. Depletion of Hdac6 expression in the CF mice (CF/Hdac6) significantly improves these responses to more WT levels. Weight loss in response to infection is most severe in CF mice and significantly attenuated in CF/Hdac6 mice. Bacterial levels are reduced at a faster rate in CF/Hdac6 mice compared to CF mice where infection persists. Percent neutrophils in lung lavage fluid post-infection are significantly higher in CF mice, but returned to WT levels with CF/Hdac6 mice. Similarly, CF Mip-2 levels are restored to WT levels in the absence of Hdac6 expression. These data demonstrate that Hdac6 depletion restores CF responses to bacterial challenge to WT-like profiles and offer a potential therapeutic avenue for addressing inflammation and infection in CF airways independently of Cftr correction.

High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared with respective wild-type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule-dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK inhibitor compound C. We conclude that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. These findings define a potential mechanism to explain the efficacy of ibuprofen therapy in CF.

Growth failure in cystic fibrosis (CF) patients has been well-documented and shown to correlate with poorer disease outcomes. This observation is also true in CF animal models, including mouse, pig, rat, and ferret. The etiology underlying growth deficits is unknown, and our previous work demonstrated reduced tubulin acetylation in CF cell models and tissue that is correctable by inhibition of histone deacetylase-6 (HDAC6). Here, we hypothesize that loss of HDAC6 will improve growth phenotype in a CF mouse model. Hdac6 knockout mice were crossed with F508del (CF) mice to generate F508del/Hdac6 (CF/HDA) mice. Growth, fat deposits, survival, and bioelectric measurements were analyzed. CF/HDA mice displayed improvements in length and weight with no correction of CFTR function. Mechanistically, Igf1 levels likely account for increased length and improvements in fertility. Weight gain is attributed to increased fat deposits potentially mediated by increased adipocyte differentiation. CF-related growth deficits can be improved via inhibition of HDAC6, further implicating it as a potential therapeutic target for CF.