Lucie Knabe

Supplementing Defect in Club Cell Secretory Protein Attenuates Airway Inflammation in COPD

BACKGROUND Club cell secretory protein (CCSP) is a protective biomarker associated with annual decline in lung function. COPD progression results from an imbalance between injury and repair initially triggered by cigarette smoking. OBJECTIVE We investigated the effect of CCSP as a therapeutic strategy to restore the balance between injury and repair in COPD simultaneously, validating an ex vivo air-liquid interface (ALI) culture of human bronchial epithelial cells. METHODS Endobronchial biopsy specimens (EBBs) were obtained from 13 patients with COPD, eight smokers, and eight control subjects. Morphometric analysis of the initial EBBs was performed. ALI cultures derived from the same EBBs were exposed to cigarette smoke extract (CSE) with or without exogenous recombinant human CCSP (rhCCSP) supplementation. CCSP and IL-8 concentrations were assessed at steady state and after CSE exposure. RESULTS Morphometric analysis of the initial EBBs showed increased cell density but decreased immunostaining of CCSP+ cells in EBBs of patients with COPD (P = .03 vs control subjects). At steady state, lower CCSP (P = .04) and higher IL-8 levels (P < .0001) were found in COPD ALI epithelium. Exogenous rhCCSP supplementation dampened CSE-induced IL-8-release in patients with COPD and returned to levels similar to those of smokers and control subjects (P = .0001). A negative correlation was found between IL-8-release in ALI and CCSP+ cell density in initial biopsy specimens (P = .0073). CONCLUSIONS In vitro, rhCCSP exogenous supplementation can reverse CSE-induced IL-8 release in biopsy specimens from patients with COPD, indicating a potential use of this strategy in vivo.

Curative diet supplementation with a melon superoxide dismutase reduces adipose tissue in obese hamsters by improving insulin sensitivity

SCOPE Obesity-related metabolic syndrome is often associated with a decrease of insulin sensitivity, inducing several modifications. However, dietary antioxidants could prevent insulin resistance. We have previously shown the preventive effects of a melon superoxide dismutase (SOD) in obese hamsters. However, its antioxidant effects have never been studied on adipose tissue. METHODS AND RESULTS We evaluated the effects of a 1-month curative supplementation with SODB on the adipose tissue of obese hamsters. Animals received either a standard diet or a cafeteria diet for 15 wk. Cafeteria diet induced obesity and related disorders, including insulin resistance and oxidative stress, in the abdominal adipose tissue. After SODB supplementation, the adipose tissue weight was decreased, probably by activating adipocytes lipolysis and thus reducing their size. SODB treatment also resulted in abdominal adipose tissue fibrosis reduction. Finally, SODB administration increased the expression of endogenous antioxidant enzymes and thus reduced oxidative stress and insulin resistance. The improvement of insulin sensitivity observed after SODB treatment could explain adipocyte lipolysis activation and fibrosis reduction. CONCLUSION These findings demonstrate that a dietary SOD supplementation could be a useful strategy against obesity-related modifications in adipose tissue.

Club cells and CC16: another “smoking gun”? (With potential bullets against COPD)

Chronic obstructive pulmonary disease (COPD) remains a challenge for clinicians and scientists, as no currently available treatment can significantly change the natural history of the disease. Accelerated lung function decline and recurrent episodes of exacerbations are thought to play critical roles in precipitating patients towards comorbid conditions, long-term oxygen supplementation dependency, and finally death. Currently, the underlying paradigm counterbalances alveolar wall destruction with peribronchiolar fibrosis, the latter being hypothesised to precede the former [1]. Although small airways are now largely recognised as the critical zone of disease initiation and progression [2], very few histological and biological properties specific to this compartment in humans have been highlighted to explain this phenomenon. Greater susceptibility of knockout CC16 mice to cigarette smoking gives insight regarding a potential player in COPD http://ow.ly/KhCMa

Epithelial ciliated beating cells essential for ex vivo ALI culture growth

BackgroundBronchial epithelium plays a key role in orchestrating innate and adaptive immunity. The fate of ex vivo airway epithelial cultures growing at the air liquid interface (ALI) derived from human endobronchial biopsies or brushings is not easy to predict. Calibrating and differentiating these cells is a long and expensive process requiring rigorous expertise. Pinpointing factors associated with ALI culture success would help researchers gain further insight into epithelial progenitor behavior.MethodsA successful ALI culture was defined as one in which a pseudostratified epithelium has formed after 28 days in the presence of all differentiated epithelial cell types. A 4-year prospective bi-center study was conducted with adult subjects enrolled in different approved research protocols.Results463 consecutive endobronchial biopsies were obtained from normal healthy volunteers, healthy smokers, asthmatic patients and smokers with COPD. All demographic variables, the different fiber optic centers and culture operators, numbers of endo-bronchial biopsies and the presence of ciliated cells were carefully recorded. Univariate and multivariate models were developed. A stepwise procedure was used to select the final logistic regression model. ALI culture success was independently associated with the presence of living ciliated cells within the initial biopsy (OR = 2.18 [1.50–3.16], p < 0.001).ConclusionThis finding highlights the properties of the cells derived from the epithelium dedifferentiation process. The preferential selection of samples with ciliated beating cells would probably save time and money. It is still unknown whether successful ALI culture is related to indicators of general cell viability or a purported stem cell state specifically associated with ciliated beating cells.

The HDAC inhibitor SAHA does not rescue CFTR membrane expression in Cystic Fibrosis

The development of suitable Cystic Fibrosis (CF) models for preclinical bench tests of therapeutic candidates is challenging. Indeed, the validation of molecules to rescue the p.Phe508del-CFTR channel (encoded by the Cystic Fibrosis Transmembrane conductance Regulator gene carrying the p.Phe508del mutation) requires taking into account their overall effects on the epithelium. Suberoylanilide Hydroxamic Acid (SAHA), a histone deacetylase inhibitor (HDACi), was previously shown to be a CFTR corrector via proteostasis modulation in CFTR-deficient immortalized cells. Here, we tested SAHA effects on goblet cell metaplasia using an ex vivo model based on the air-liquid interface (ALI) culture of differentiated airway epithelial cells obtained by nasal scraping from CF patients and healthy controls. Ex vivo epithelium grew successfully in ALI cultures with significant rise in the expression of CFTR and of markers of airway epithelial differentiation compared to monolayer cell culture. SAHA decreased CFTR transcript and protein levels in CF and non-CF epithelia. Whereas SAHA induced lysine hyperacetylation, it did not change histone modifications at the CFTR promoter. SAHA reduced MUC5AC and MUC5B expression and inhibited goblet epithelial cell differentiation. Similar effects were obtained in CF and non-CF epithelia. All the effects were fully reversible within five days from SAHA withdrawal. We conclude that, ex vivo, SAHA modulate the structure of airway epithelia without specific effect on CFTR gene and protein suggesting that HDACi cannot be useful for CF treatment.

Club cell secretory protein serum concentration is a surrogate marker of small-airway involvement in asthmatic patients

Poor asthma control and recurrent exacerbations have been shown to be a phenotypic counterpart of asthma with predominantly small-airway involvement.1 Biomarkers are not always accurate in asthmatic patients, especially in serum, because compartmentalization can occur between the blood and airways. Blood eosinophil counts do not represent an overall view of airway inflammation, and exhaled nitric oxide measurements at different flow rates (fraction of exhaled nitric oxide [Feno] and alveolar nitric oxide [Calvno]) have been developed and validated to reflect more accurately proximal and distal airway inflammation.2 Club cell secretory protein (CCSP) serum concentration has been shown to be associated with chronic obstructive pulmonary disease, bronchiolitis obliterans syndrome, and sarcoidosis, which are all predominantly diseases involving the small airways. Ranges of CCSP concentrations in healthy subjects, reproducibility, and relationships between serum and airway levels are known and can be used as potential surrogate markers. Our aim was to assess small-airway disease in asthmatic patients and to find a related biomarker. We used a dynamic assessment of gas trapping using computed tomographic (CT) imaging of the chest during methacholine challenge as a marker of small-airway disease.

LIFE BEYOND LIFE – An Easy Way to Derive Lung Fibroblasts from Cadavers

Several protocols have illustrated the possibility of deriving cells, such as fibroblasts, from different organs. These techniques generally concern organs sampled from living persons, but have already been described for cadavers, especially concerning the skin and tendons. We present, for the first time, an easy way to derive pulmonary fibroblasts from a lung tissue sampled from a cadaver and directly culture plated. The fibroblast output was checked daily. We obtained lung fibroblasts from 3 (60%) cadavers and 2 (100%) living persons. The fibroblast output took about 3 days for cells from living persons and took up to 39 days for those from cadavers. We did not clearly identify any parameters that could explain these differences. Nevertheless, these derived cells had the same features as the source cells, especially in terms of morphology and proliferation, and could potentially be used in different research domains such as forensic or regeneration medicine.

CCSP G38A polymorphism environment interactions regulate CCSP levels differentially in COPD

Impaired airway homeostasis in chronic obstructive pulmonary disease (COPD) could be partly related to club cell secretory protein (CCSP) deficiency. We hypothesize that CCSP G38A polymorphism is involved and aim to examine the influence of the CCSP G38A polymorphism on CCSP transcription levels and its regulatory mechanisms. CCSP genotype and CCSP levels in serum and sputum were assessed in 66 subjects with stable COPD included in a 1-yr observational study. Forty-nine of them had an exacerbation. In an in vitro study, the impact on the CCSP promoter of 38G wild-type or 38A variant was assessed. BEAS-2B cells were transfected by either the 38G or 38A construct, in the presence/absence of cigarette smoke extract (CSE) or lipopolysaccharides (LPS). Cotransfections with modulating transcription factors, p53 and Nkx2.1, identified by in silico analysis by using ConSite and TFSEARCH were conducted. A allele carrier COPD patients had lower serum and sputum CCSP levels, especially among active smokers, and a decreased body mass index, airflow obstruction, dyspnea, and exercise capacity (BODE) score. In vitro, baseline CCSP transcription levels were similar between the wild and variant constructs. CSE decreased more profoundly the CCSP transcription level of 38A transfected cells. The opposite effect was observed with p53 cotransfection. LPS stimulation induced CCSP repression in 38A promoter transfected cells. Cotransfection with Nkx2.1 significantly activated the CCSP promoters irrespective of the polymorphism. Circulating CCSP levels are associated with smoking and the CCSP G38A polymorphism. CSE, LPS, and the Nkx2.1 and p53 transcription factors modulated the CCSP promoter efficiency. The 38A polymorphism exaggerated the CCSP repression in response to p53 and CSE.