Neutrophil elastase-mediated increase in airway temperature during inflammation  Annika Schmidt, Azzaq Belaaouaj, Rosi Bissinger, Garrit Koller, Laurette Malleret, Ciro D'Orazio, Martino Facchinelli, Bernhard Schulte- Hubbert, Antonio Molinaro, Otto Holst, Jutta Hammermann, Monika Schniederjans, Keith C. Meyer, Soeren Damkiaer, Giorgio Piacentini, Baroukh Assael, Kenneth Bruce, Susanne Häußler, John J. LiPuma, Joachim Seelig, Dieter Worlitzsch, Gerd Döring  Journal of Cystic Fibrosis  Volume 13, Issue 6, Pages 623-631 (December 2014) DOI: 10.1016/j.jcf.2014.03.004 Copyright © 2014 European Cystic Fibrosis Society. Terms and Conditions

Fig. 1 Neutrophil elastase (NE)/α1-proteinase inhibitor (α1-PI) complex formation increases local temperature during infection/inflammation. A, B, exhaled breath temperature correlates with lung function in CF patients. Exhaled breath temperature was determined in 56 CF patients (red) and 20 healthy individuals (black) using the end-expiratory manoeuver plateau temperature (PLET) method. Forced expiratory volume in 1s was determined by spirometry. The Student's t-test was used for statistical evaluation. C, local temperature in neutrophil-dominated mucus plugs is increased above airway luminal temperature and normal body temperature in CF patients. For in vivo temperature measurements in airways of 5 CF patients and 3 individuals with bronchial carcinoma, a computerized Clark type oxygen probe was inserted in the working channel of the bronchoscope and guided under video control into the right upper lobe where measurements were made in mucopurulent material and in the airway lumen. D, E, NE/α1-PI complex formation is exothermic. The enthalpy of the complex formation between NE and α1-PI was determined by isothermal titration calorimetry (ITC). The calorimeter cell (Vcell=0.203ml) contained NE at a concentration of 15μM. The syringe contained α1-PI at a concentration of 150μM. Measurement performed at 35°C in 150mM NaCl, 10mM sodium phosphate, pH7.4. (D) Heat flow. Each exothermic peak corresponds to the injection of 2μL α1-PI solution into the calorimeter cell. (E) Binding enthalpy as a function of the molar ratio α1-PI/NE. ■: experimental data; solid line: theoretical prediction based on a 1:1 binding model of NE with α1-PI using a binding constant of K=5×107M−1 and a binding enthalpy of ΔH0=−18.5kcal/mol. F, Pouch airspace temperature in wild type mice is increased compared with that in neutrophil elastase-deficient (NE−/−) mice. In groups of 5 NE−/− mice air pouches were generated which were challenged with 107CFU/pouch of the P. aeruginosa strain H103. After 4days, the temperature in the pouch airspace was determined (for details see text). G, H, Total cell counts do not differ in pouches of wild type and NE−/− mice. Pouches were lavaged with PBS, and total and differential counts were performed immediately by routing methods using cytospins and morphologic analyses. Polymorphnuclear cells (PMN): WT vs NE−/−: p=0.40; Mononuclear cells (MNC): WT vs NE−/−: p=0.43. Journal of Cystic Fibrosis 2014 13, 623-631DOI: (10.1016/j.jcf.2014.03.004) Copyright © 2014 European Cystic Fibrosis Society. Terms and Conditions

Fig. 2 P. aeruginosa grown anaerobically at an elevated temperature. A, The P. aeruginosa strain PAO1 was grown in liquid media at 30°C, 37°C, 38°C and 39°C under anaerobic conditions for 96h and bacterial CFU were determined using routine methods. B, At 39°C the P. aeruginosa strain PAO1 and the alginate overexpressing mutant PAO1 SD4 (∆ mucA) were significantly compromised compared to the nonmucoid strain PAO1 SD40 (∆ mucA/algT) under anaerobic conditions after 96h incubation. Journal of Cystic Fibrosis 2014 13, 623-631DOI: (10.1016/j.jcf.2014.03.004) Copyright © 2014 European Cystic Fibrosis Society. Terms and Conditions

Fig. 3 P. aeruginosa does not produce a polysaccharide O-antigen chain when grown at 38°C under anaerobic conditions. The LPS fractions of P. aeruginosa PAO1 and an environmental P. aeruginosa isolate, grown at 30°C and 38°C aerobically or anaerobically, were extracted from whole cell lysate and subjected to SDS gel electrophoresis. LPS analysis reveals lack of the O-side chain at 38°C under anaerobic growth. Journal of Cystic Fibrosis 2014 13, 623-631DOI: (10.1016/j.jcf.2014.03.004) Copyright © 2014 European Cystic Fibrosis Society. Terms and Conditions