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Defective CFTR Cl− channel enhances proteasomal degradation and reduces NF-κB activity in lung epithelial cells exposed to oxidative stress
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6. Immunology
205 A transcriptome analysis of the in vitro response of Pseudomonas aeruginosa to polymorphonuclear leukocyte exposure M. Alhede1 , T. Bjarnsholt1 , P.O. Jensen2 , M. Parsek3 , N. Høiby2 , T.B. Rasmussen4 , M. Givskov1 . 1 Bioscience and Technology, Technical University of Denmark, Lyngby, Denmark; 2 Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark; 3 School of Medicine, University of Washington, Seattle, WA, USA; 4 Genomic & Strain Development, Chr. Hansen, Copenhagen, Denmark Polymorphonuclear leukocytes (PMNs) are a central part of the human innate immunity and they are dominating the response against bacterial infections in the cystic fibrosis (CF) lung. However, the opportunistic pathogen Pseudomonas aeruginosa can evade destruction by PMNs and may thus cause persistent infections in immuno-compromised patients. In the present in vitro study, interactions between P. aeruginosa (in the sessile and planktonic modes of growth) and PMNs were analyzed by means of DNA microarray based transcriptomics. We found that the P. aeruginosa wild type biofilms, in contrast to planktonic cultures and quorum sensing (QS) deficient strains, respond to PMN exposure in a rather aggressive manner. The response does not involve protective mechanisms such as those involved in oxidative stress. Rather it is dominated by an up-regulation of QS controlled virulence determinants such as those encoded by pqs, phz, rhlAB, lecB and lasB, all of which are able to cripple and eliminate eukaryotic cells. Our data supports a model by which P. aeruginosa recognizes the presence of PMN’s and channels this information through the QS system. Consequently, it strengthens the view that QS plays a key role in P. aeruginosa recognition and evasion of components of the host defense.
206 Defective CFTR Cl− channel enhances proteasomal degradation and reduces NF-úB activity in lung epithelial cells exposed to oxidative stress E. Boncoeur1 , T. Roque1 , E. Bonvin1 , V. Saint Criq1 , M. Bonora1 , A. Clement1 , O. Tabary1 , J. Jacquot1 . 1 Inserm UMR-S 719, Hˆopital St-Antoine, UPMC-Paris6, Paris, France Background: In CF patients, the loss of functional cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein in lung epithelial cells leads to reduced volume and increased oxidation of the airway surface liquid. Objective: In this study, we evaluated the role of CFTR Cl− channel activity in the control of ubiquitin-proteasome activity and NF-úB/IúB-a signaling in lung epithelial cells exposed to oxidative stress. Methods: We exposed mice for 64-h to hyperoxia-mediated oxidative stress and CF or non-CF bronchial epithelial cells during 24 h. Results: After oxidative stress, CFTR deficient mice exhibited an absence of degradation of NF-úB inhibitor (IúB-a) compared to CFTR+/ + animals. This result was accompanied by a significantly higher lung proteasomal activity. After oxidative stress, human CFTR-deficient bronchial epithelial cells exhibited higher proteasomal activity and a lack of increased NF-úB-dependent transcriptional activity compared to CFTR-sufficient lung epithelial cells. In response to oxidative stress, degradation of NF-úB inhibitor IúB-a was defective in two CFTR deficient lung models. Moreover, the inhibition of CFTR Cl− channel activity in normal bronchial immortalized cell line 16HBE14o- by a specific CFTRinh−172 inhibitor exposed to oxidative stress, caused a significant increase of proteasomal degradation activity. Conclusion: All together, our data show that functional CFTR Cl− channel activity is crucial in regulating lung proteasomal degradation and NF-úB activity in conditions of oxidative stress. Supported by: grants from Inserm, FRM and the French cystic fibrosis association (VLM).
207 Targeting Cx43 protects against lung inflammation M.Z. Richani Sarieddine1 , I. Garcia2 , B. Foglia1 , B.R. Kwak3 , M. Chanson1 . 1 Pediatrics, Geneva University Hospitals, Geneva, Switzerland; 2 Pathology and Immunology, Faculty of Medicine, Geneva, Switzerland; 3 Foundation for Medical Research, Geneva University Hospitals, Geneva, Switzerland Cystic fibrosis is a severe inflammatory disease characterized by a massive recruitment of neutrophils to the lungs. Gap junctions may serve as a conducting pathway to enhance such recruitment and spread inflammation within the lung alveoli. Indeed, we found that intratracheal instillation of 43Gap26, a blocking peptide of the gap junction protein connexin43 (Cx43), reduced inflammation by 70% in a mouse model subjected to intratracheal instillation of Pseudomonas aeruginosa LPS. In comparison to wild type animals, mice heterozygous for the Cx43 gene exhibited a lower inflammatory response, representing a 54% protection to inflammation. In contrast, an exaggerated inflammatory response characterized with a massive neutrophil recruitment was observed in a mouse model expressing a non regulatable mutant of Cx43. Finally, Cx43 expression was unaffected in mice protected from LPS-induced inflammation by inactivation of either the TNF-alpha gene or the protein. Together, our findings indicate that functional expression of Cx43 in lungs is activated by mediators of the inflammatory cascade and that this regulation modulates in turn the recruitment of neutrophils and therefore the intensity of lung inflammation. This reveals a pro-inflammatory role of Cx43, which may represent a pharmacological target to modulate the severity of lung diseases such as cystic fibrosis. Supported by: Vaincre la Mucoviscidose and the Swiss Science Foundation.
208* Immunomodulatory properties of vitamin D in Scandinavian cystic fibrosis patients T. Pincikova1 , K. Nilsson1 , F. Karpati1 , L. Hjelte1 . 1 Karolinska Institutet, Karolinska Univ Hosp, Stockholm, Sweden For the Scandinavian Study Consortium Immunosuppressive effects of vitamin (vit.) D and a potential role of vitamin D deficiency in the pathophysiology of autoimmune diseases have been described. We investigated if vitamin D deficiency could contribute to the chronic proinflammatory state and the higher occurrence of immune-hyperreactivity-related conditions in CF patients. Methods: Cross-sectionally determined vit.D variables were compared to lung function, inflammatory parameters and immune hyperreactivity markers in 898 CF patients by multiple linear regression analysis. Vit.D intake was based on a 7-day dietary food record. Findings: Blood vit.D positively correlated with FEV1 (p < 0.01) as well as with FVC (p < 0.01). Negative correlation was found between blood vit.D and IgG (p < 0.001), supplemented vit.D per kg BW and IgG (p < 0.001), total vit.D intake per kg BW and IgG (p < 0.05). Additionally, lower blood vitamin D levels were related to both presence of pathological OGTT (p < 0.05) and occurrence of CFRD (p < 0.05). Correspondingly, total vitamin D intake (p < 0.01) and total vitamin D intake per kg BW (p < 0.01) correlated negatively with HbA1C values. In children (n = 442) blood vitamin D correlated negatively with transglutaminase levels (p < 0.05). Average vitamin D concentrations did not correlate with the antiinflammatory cytokine IL-10 in a limited series. Conclusion: Vitamin D deficiency might contribute to the continuous shift towards inflammation as well as to the higher prevalence of DM and celiac disease in CF patients. New recommendations for vitamin D supplementation and monitoring of blood vitamin D levels are needed. Supported by: Swedish Heart Lung Foundation, Stiftelsen Frimurare-Barnhuset i Stockholm, Norwegian and Swedish CF Associations, and by an unrestricted grant from Solvay Pharma.
6. Immunology
205 A transcriptome analysis of the in vitro response of Pseudomonas aeruginosa to polymorphonuclear leukocyte exposure M. Alhede1 , T. Bjarnsholt1 , P.O. Jensen2 , M. Parsek3 , N. Høiby2 , T.B. Rasmussen4 , M. Givskov1 . 1 Bioscience and Technology, Technical University of Denmark, Lyngby, Denmark; 2 Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark; 3 School of Medicine, University of Washington, Seattle, WA, USA; 4 Genomic & Strain Development, Chr. Hansen, Copenhagen, Denmark Polymorphonuclear leukocytes (PMNs) are a central part of the human innate immunity and they are dominating the response against bacterial infections in the cystic fibrosis (CF) lung. However, the opportunistic pathogen Pseudomonas aeruginosa can evade destruction by PMNs and may thus cause persistent infections in immuno-compromised patients. In the present in vitro study, interactions between P. aeruginosa (in the sessile and planktonic modes of growth) and PMNs were analyzed by means of DNA microarray based transcriptomics. We found that the P. aeruginosa wild type biofilms, in contrast to planktonic cultures and quorum sensing (QS) deficient strains, respond to PMN exposure in a rather aggressive manner. The response does not involve protective mechanisms such as those involved in oxidative stress. Rather it is dominated by an up-regulation of QS controlled virulence determinants such as those encoded by pqs, phz, rhlAB, lecB and lasB, all of which are able to cripple and eliminate eukaryotic cells. Our data supports a model by which P. aeruginosa recognizes the presence of PMN’s and channels this information through the QS system. Consequently, it strengthens the view that QS plays a key role in P. aeruginosa recognition and evasion of components of the host defense.
206 Defective CFTR Cl− channel enhances proteasomal degradation and reduces NF-úB activity in lung epithelial cells exposed to oxidative stress E. Boncoeur1 , T. Roque1 , E. Bonvin1 , V. Saint Criq1 , M. Bonora1 , A. Clement1 , O. Tabary1 , J. Jacquot1 . 1 Inserm UMR-S 719, Hˆopital St-Antoine, UPMC-Paris6, Paris, France Background: In CF patients, the loss of functional cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein in lung epithelial cells leads to reduced volume and increased oxidation of the airway surface liquid. Objective: In this study, we evaluated the role of CFTR Cl− channel activity in the control of ubiquitin-proteasome activity and NF-úB/IúB-a signaling in lung epithelial cells exposed to oxidative stress. Methods: We exposed mice for 64-h to hyperoxia-mediated oxidative stress and CF or non-CF bronchial epithelial cells during 24 h. Results: After oxidative stress, CFTR deficient mice exhibited an absence of degradation of NF-úB inhibitor (IúB-a) compared to CFTR+/ + animals. This result was accompanied by a significantly higher lung proteasomal activity. After oxidative stress, human CFTR-deficient bronchial epithelial cells exhibited higher proteasomal activity and a lack of increased NF-úB-dependent transcriptional activity compared to CFTR-sufficient lung epithelial cells. In response to oxidative stress, degradation of NF-úB inhibitor IúB-a was defective in two CFTR deficient lung models. Moreover, the inhibition of CFTR Cl− channel activity in normal bronchial immortalized cell line 16HBE14o- by a specific CFTRinh−172 inhibitor exposed to oxidative stress, caused a significant increase of proteasomal degradation activity. Conclusion: All together, our data show that functional CFTR Cl− channel activity is crucial in regulating lung proteasomal degradation and NF-úB activity in conditions of oxidative stress. Supported by: grants from Inserm, FRM and the French cystic fibrosis association (VLM).
207 Targeting Cx43 protects against lung inflammation M.Z. Richani Sarieddine1 , I. Garcia2 , B. Foglia1 , B.R. Kwak3 , M. Chanson1 . 1 Pediatrics, Geneva University Hospitals, Geneva, Switzerland; 2 Pathology and Immunology, Faculty of Medicine, Geneva, Switzerland; 3 Foundation for Medical Research, Geneva University Hospitals, Geneva, Switzerland Cystic fibrosis is a severe inflammatory disease characterized by a massive recruitment of neutrophils to the lungs. Gap junctions may serve as a conducting pathway to enhance such recruitment and spread inflammation within the lung alveoli. Indeed, we found that intratracheal instillation of 43Gap26, a blocking peptide of the gap junction protein connexin43 (Cx43), reduced inflammation by 70% in a mouse model subjected to intratracheal instillation of Pseudomonas aeruginosa LPS. In comparison to wild type animals, mice heterozygous for the Cx43 gene exhibited a lower inflammatory response, representing a 54% protection to inflammation. In contrast, an exaggerated inflammatory response characterized with a massive neutrophil recruitment was observed in a mouse model expressing a non regulatable mutant of Cx43. Finally, Cx43 expression was unaffected in mice protected from LPS-induced inflammation by inactivation of either the TNF-alpha gene or the protein. Together, our findings indicate that functional expression of Cx43 in lungs is activated by mediators of the inflammatory cascade and that this regulation modulates in turn the recruitment of neutrophils and therefore the intensity of lung inflammation. This reveals a pro-inflammatory role of Cx43, which may represent a pharmacological target to modulate the severity of lung diseases such as cystic fibrosis. Supported by: Vaincre la Mucoviscidose and the Swiss Science Foundation.
208* Immunomodulatory properties of vitamin D in Scandinavian cystic fibrosis patients T. Pincikova1 , K. Nilsson1 , F. Karpati1 , L. Hjelte1 . 1 Karolinska Institutet, Karolinska Univ Hosp, Stockholm, Sweden For the Scandinavian Study Consortium Immunosuppressive effects of vitamin (vit.) D and a potential role of vitamin D deficiency in the pathophysiology of autoimmune diseases have been described. We investigated if vitamin D deficiency could contribute to the chronic proinflammatory state and the higher occurrence of immune-hyperreactivity-related conditions in CF patients. Methods: Cross-sectionally determined vit.D variables were compared to lung function, inflammatory parameters and immune hyperreactivity markers in 898 CF patients by multiple linear regression analysis. Vit.D intake was based on a 7-day dietary food record. Findings: Blood vit.D positively correlated with FEV1 (p < 0.01) as well as with FVC (p < 0.01). Negative correlation was found between blood vit.D and IgG (p < 0.001), supplemented vit.D per kg BW and IgG (p < 0.001), total vit.D intake per kg BW and IgG (p < 0.05). Additionally, lower blood vitamin D levels were related to both presence of pathological OGTT (p < 0.05) and occurrence of CFRD (p < 0.05). Correspondingly, total vitamin D intake (p < 0.01) and total vitamin D intake per kg BW (p < 0.01) correlated negatively with HbA1C values. In children (n = 442) blood vitamin D correlated negatively with transglutaminase levels (p < 0.05). Average vitamin D concentrations did not correlate with the antiinflammatory cytokine IL-10 in a limited series. Conclusion: Vitamin D deficiency might contribute to the continuous shift towards inflammation as well as to the higher prevalence of DM and celiac disease in CF patients. New recommendations for vitamin D supplementation and monitoring of blood vitamin D levels are needed. Supported by: Swedish Heart Lung Foundation, Stiftelsen Frimurare-Barnhuset i Stockholm, Norwegian and Swedish CF Associations, and by an unrestricted grant from Solvay Pharma.