Guillaume F. Bouvet

Induction of nitric oxide synthase expression by lipopolysaccharide is mediated by calcium-dependent PKCα-β1 in alveolar epithelial cells

Nitric oxide (NO) plays an important role in innate host defense and inflammation. In response to infection, NO is generated by inducible nitric oxide synthase (iNOS), a gene product whose expression is highly modulated by different stimuli, including lipopolysaccharide (LPS) from gram-negative bacteria. We reported recently that LPS from Pseudomonas aeruginosa altered Na⁺ transport in alveolar epithelial cells via a suramin-dependent process, indicating that LPS activated a purinergic response in these cells. To further study this question, in the present work, we tested whether iNOS mRNA and protein expression were modulated in response to LPS in alveolar epithelial cells. We found that LPS induced a 12-fold increase in iNOS mRNA expression via a transcription-dependent process in these cells. iNOS protein, NO, and nitrotyrosine were also significantly elevated in LPS-treated cells. Ca²⁺ chelation and protein kinase C (PKCα-β1) inhibition suppressed iNOS mRNA induction by LPS, implicating Ca²⁺-dependent PKC signaling in this process. LPS evoked a significant increase of extracellular ATP. Because PKC activation is one of the signaling pathways known to mediate purinergic signaling, we evaluated the hypothesis that iNOS induction was ATP dependent. Although high suramin concentration inhibited iNOS mRNA induction, the process was not ATP dependent, since specific purinergic receptor antagonists could not inhibit the process. Altogether, these findings demonstrate that iNOS expression is highly modulated in alveolar epithelial cells by LPS via a Ca²⁺/PKCα-β1 pathway independent of ATP signaling.

Combined Exercise Training Improves Glycemic Control in Adult With Cystic Fibrosis.

Purpose Glucose abnormality and diabetes are the most common comorbidities in cystic fibrosis (CF). Combined (aerobic and resistance) exercise program in type 2 patients with diabetes demonstrated an improvement of glycemic control. The aim of the study was to determine whether a combined exercise program is beneficial to improve plasma glucose at 2 h of the oral glucose tolerance test in CF. Method Eighteen adults with CF with glucose abnormality were recruited (Clinicaltrial.gov: NTC02127957), and 17 were randomly assigned to a control or exercise group for 12 wk. V˙O2max, oral glucose tolerance test, muscular endurance and strength, and quality of life were measured pre- and postintervention. Results Fourteen participants completed the protocol. Patients in the exercise group improved significantly their 2-h plasma glucose values (−2.34 ± 1.26 mmol·L−1, P < 0.007, confidence interval = 99.22%) and presented a reduction of 17.2% (P < 0.05) in total glucose excursion. No significant change for other parameters was observed. Conclusion A combined exercise program improves glycemic control in CF.

Association between serum YKL-40 level and dysglycemia in cystic fibrosis.

BACKGROUND YKL-40, a chitinase-like protein, is a biomarker for type 1 and type 2 diabetes prognosis. We hypothesized that YKL-40 protein levels are elevated in CF patients with dysglycemia. METHODS Seventeen healthy control subjects and 66 CF patients were prospectively recruited and subjected to an oral glucose tolerance test. In all participants, fasting serum YKL-40 was compared between control and CF patients and between normal glucose-tolerant patients (NG-CF) and CF patients with dysglycemia (DG-CF). A Botnia clamp procedure was performed on a subset of patients for each group to determine the impact of acute increases of either glucose or insulin on YKL-40 concentration. RESULTS CF patients had higher serum YKL-40 values than the controls (113 [49;288] vs. 38 [30;50] ng/ml, p<0.001). YKL-40 concentrations in CF patients were mainly increased in the DG-CF group, who had significantly higher values: 213 [93;383] vs. 67 [27;97] ng/ml in the NG-CF group, p<0.001). No significant modulation of YKL-40 concentration was observed in serum of CF (NG or DG-CF) or non-CF patients, after acute exposure to glucose or insulin. CONCLUSIONS Higher serum YKL-40 levels in CF patients are significantly associated with dysglycemia. The increase in YKL-40 is potentially associated with an inflammatory response resulting from chronic glucose intolerance or CF disease evolution.

Oxidative stress modulates the expression of genes involved in cell survival in ΔF508 cystic fibrosis airway epithelial cells.

Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 μM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared with NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients.

DNA Methylation Regulates RGS2-induced S100A12 Expression in Airway Epithelial Cells

&NA; RGS2 is a key modulator of stress in human airway epithelial cells, especially of hyperresponsiveness and mucin hypersecretion, both of which are features of cystic fibrosis (CF). Because its expression can be modulated through the DNA methylation pathway, we hypothesize that RGS2 is downregulated by DNA hypermethylation in CF airway epithelial cells. This downregulation would then lead to an enhanced inflammatory response. We demonstrated RGS2 transcript and protein downregulation in cultured airway epithelial cells from patients with CF and validated our findings in two CF epithelial cell lines. A methylated DNA immunoprecipitation array showed the presence of methylated cytosine on 13 gene promoters in CF. Among these genes, we confirmed that the RGS2 promoter was hypermethylated by using bisulfite conversion coupled with a methylation‐specific PCR assay. Finally, we showed that downregulation of RGS2 in non‐CF cells increased the expression of S100A12, a proinflammatory marker. These results highlight the importance of epigenetic regulation in gene expression in CF and show that RGS2 might modulate the inflammatory response in CF through DNA methylation control.

Proprotein Convertase Subtilisin/Kexin type 9 affects insulin but not lipid metabolism in cystic fibrosis

PURPOSE Cystic Fibrosis (CF) is the most common genetic disorder and, with improved survival, glucose abnormalities have emerged as a major comorbidity. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a regulator of plasma LDL-cholesterol homeostasis, is associated with lipid and glucose metabolism in healthy individuals. Here we report on the link between PCSK9 and markers of metabolism in CF. METHODS Cross-sectional analysis was performed on CF patients (≥ 18 years, N=94) from the Montreal Cohort, without known diabetes, and on healthy individuals (N=19). The levels of PCSK9 and lipid markers were quantified and all subjects underwent a 2 h oral glucose tolerance test. RESULTS No significant differences in PCSK9 levels were found between healthy individuals and patients with CF, or between the groups with different degrees of glucose tolerance. No association was found between PCSK9 and markers of lipid metabolism; however, a positive correlation was found between PCSK9 and total insulin secretion and a negative one with insulin sensitivity in CF patients who had normal glucose tolerance. CONCLUSION Circulating levels of PCSK9 in the CF population are comparable to those in the healthy population. There are no associations between PCSK9 levels and either glucose or lipid homeostasis parameters. Nevertheless, a statistically significant link was observed between PCSK9 and markers of insulin homeostasis, solely in CF patients who presented normal glucose tolerance. Further exploration of the relationship between PCSK9 and insulin homeostasis in CF patients with normal glucose tolerance is warranted.

Undiagnosed Chronic Granulomatous Disease, Burkholderia cepacia complex Pneumonia, and Acquired Hemophagocytic Lymphohistiocytosis: A Deadly Association

Background. Chronic granulomatous disease is a rare inherited disorder of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The clinical course of the disease is marked by recurrent infections, including Burkholderia cepacia complex infection. Case Report. Here we report the case of a 21-year-old male hospitalized for a Burkholderia cepacia complex pneumonia. Despite the broad spectrum antibiotic treatment, fever continued and patients condition worsened. Anemia and thrombocytopenia developed together with hypofibrinogenemia. The patient died of multiple organ dysfunction 17 days after his admission. Autopsy revealed hemophagocytosis, suggesting the diagnosis of acquired hemophagocytic lymphohistiocytosis. DNA analysis showed a deletion in the p47phox gene, confirming the diagnosis of autosomal recessive chronic granulomatous disease. Discussion. In addition to chronic granulomatous disease, recent findings have demonstrated that Burkholderia cepacia complex can decrease activity of the NADPH oxidase. Interestingly, hemophagocytic lymphohistiocytosis is characterized by an impaired function of the T-cell mediated inflammation which is partly regulated by the NADPH oxidase. Physicians should therefore pay particular attention to this deadly association.