JCF-01033; No of Pages 9

Journal of Cystic Fibrosis xx (2014) xxx – xxx www.elsevier.com/locate/jcf

Original Article

Neutrophil elastase-mediated increase in airway temperature during inflammation Annika Schmidt a,1 , Azzaq Belaaouaj b,1 , Rosi Bissinger a , Garrit Koller c , Laurette Malleret b , Ciro D'Orazio d , Martino Facchinelli e , Bernhard Schulte-Hubbert f , Antonio Molinaro g , Otto Holst h , Jutta Hammermann i , Monika Schniederjans j , Keith C. Meyer k , Soeren Damkiaer l , Giorgio Piacentini e , Baroukh Assael d , Kenneth Bruce c , Susanne Häußler h , John J. LiPuma m , Joachim Seelig n , Dieter Worlitzsch o,⁎, Gerd Döring a,1 a

h

Institute of Medical Microbiology and Hygiene, German Center for Infection Research, University Clinic Tübingen, Germany b INSERM U-1111, Centre International de Recherche en Infectiologie (CIRI), Lyon, France c King's College, London, England d Department of Pediatrics, Verona, Italy e Ospedale Civile Maggiore, Verona, Italy f Medical Clinic und Policlinic I Pneumology, Technical University Dresden, Dresden, Germany g Department of Chemical Sciences, Università di Napoli Federico II, Italy Research Center Borstel, Center for Medicine and Biosciences, Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Borstel, Germany i Department of Pediatrics, Technical University Dresden, Dresden, Germany j Helmholtz-Centre for Infection Research, Braunschweig, Germany k University of Wisconsin School of Medicine, Madison, USA l Department of Systems Biology and Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark m Department of Paediatrics, University of Michigan, Ann Arbor, USA n Biophysical Chemistry, University of Basel, Basel Switzerland o Institute of Hygiene, University of Halle, Germany Received 3 January 2014; received in revised form 13 March 2014; accepted 13 March 2014 Available online xxxx

Abstract Background: How elevated temperature is generated during airway infections represents a hitherto unresolved physiological question. We hypothesized that innate immune defence mechanisms would increase luminal airway temperature during pulmonary infection. Methods: We determined the temperature in the exhaled air of cystic fibrosis (CF) patients. To further test our hypothesis, a pouch inflammatory model using neutrophil elastase-deficient mice was employed. Next, the impact of temperature changes on the dominant CF pathogen Pseudomonas aeruginosa growth was tested by plating method and RNAseq. Results: Here we show a temperature of ~ 38 °C in neutrophil-dominated mucus plugs of chronically infected CF patients and implicate neutrophil elastase:α1-proteinase inhibitor complex formation as a relevant mechanism for the local temperature rise. Gene expression of the main pathogen in CF, P. aeruginosa, under anaerobic conditions at 38 °C vs 30 °C revealed increased virulence traits and characteristic cell wall changes.

⁎ Corresponding author at: Institute of Hygiene, University Hospital, Martin-Luther-University Halle, Magdeburger Str. 24, 06097 Halle/Saale, Germany. Tel.: +49 345 557 1103; fax: +49 345 557 1093. E-mail address: [email protected] (D. Worlitzsch). 1 A.S. and A.B. contributed equally to this work. This study is dedicated to the memory of Gerd Döring and his commitment to CF research.

http://dx.doi.org/10.1016/j.jcf.2014.03.004 1569-1993/© 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Please cite this article as: Schmidt A, et al, Neutrophil elastase-mediated increase in airway temperature during inflammation, J Cyst Fibros (2014), http://dx.doi.org/ 10.1016/j.jcf.2014.03.004

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A. Schmidt et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx

Conclusion: Neutrophil elastase mediates increase in airway temperature, which may contribute to P. aeruginosa selection during the course of chronic infection in CF. © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Neutrophil elastase; Temperature; Inflammation; Pseudomonas aeruginosa

1. Introduction Mammals maintain and closely regulate elevated core body temperature (TB) by the hypothalamus via a complex feedback system [1]. Besides their evolutionary importance [2,3], endothermy and homeothermy provide mammals with increased fitness against fungal infections over a tight range of temperature from 35.9 °C to 37.7 °C [4]. Whilst this “thermal exclusion zone” does not affect bacterial pathogens, which generally grow well at physiological temperature in mammals, the elevated human core TB provides optimal metabolic conditions for phagocytic cell activity. For instance, maximum killing of the opportunistic pathogen, Staphylococcus aureus, by human neutrophils has been observed at 36–40 °C [5]. The airways are formally considered “outside” of the body, and, therefore, it is unclear how core TB [1] is regulated in human airways, particularly during pulmonary infection. We hypothesized that innate immune defence mechanisms, activated as a consequence of bacterial lung infection, would contribute to the increase of luminal airway temperature during pulmonary infection. We tested this hypothesis in patients with the hereditary disease cystic fibrosis (CF). CF patients suffer from chronic bacterial airway infections that are characterized by a large influx of polymorphonuclear leukocytes (neutrophils), which form highly viscous mucus plugs around the bacterial pathogens that persist in the airway lumen [6,7]. Our studies revealed that the binding of neutrophil elastase (NE), to its physiologic inhibitor, α1-proteinase inhibitor (α1-PI), contributes to temperature rise in inflamed airways.

2. Methods 2.1. Patients We determined the temperature in the exhaled air of 56 CF patients, aged 6–17 years, attending the outpatient clinic of the CF centre in Verona, Italy. Patients with fever (3 times daily auricular temperature measurement N 38 °C) were excluded. In addition, we recruited 20 healthy subjects, aged 7–17 years, from the outpatient clinic of the Department of Paediatrics, Policlinico G. B. Rossi, Verona, Italy. Individuals with a previous diagnosis of asthma or another respiratory disease were excluded. All controls were free from any respiratory infection for a minimum of 3 weeks prior to the study. For bronchial temperature measurements, 5 CF patients (3 males, mean age 32.6 years, forced expiratory volume in one second % predicted, FEV1%: 63% and 2 females, mean age: 26 years, FEV1%: 40.5%)

attending the CF centre of the University of Dresden, Germany, and the CF centre of the University of Wisconsin, School of Medicine, Madison, USA, were included. Additionally, we determined the bronchial temperature in 3 non-CF individuals with diagnosis of an unknown solitary pulmonary node, attending the Medical Clinic und Policlinic I Pneumology of the Technical University Dresden. Body core temperature in the CF patients was 36.7 ± 0.4 °C, in the healthy individuals 36.5 ± 0.4 °C. Informed consent was obtained from all patients and/or parents, and all parts of the study were approved by the local ethical committees. Data, which were normally distributed, were analysed using paired Student's t test. Results with inhomogeneous variances in the Student's t test were analysed using the non-parametric Mann–Whitney test. A p value of 0.05 or less was considered significant.

2.2. Fiberoptic bronchoscopy and temperature measurements in patients Fiberoptic bronchoscopy was performed as described previously [8] with minor modifications. The exhaled breath temperature test was performed using a CareFusion respiratory function equipment (Vmax, SensorMedics,Yorba Linda, USA) [9]. A respiratory filter (Mada, Milano, Italy) was attached to the mass flow sensor for each participant to avoid microbiological contamination. Furthermore, a recently updated software application was used to increase the sensitiveness of the measurement which resulted in an intra-subject variability of b 1%, compared to 2.35% using the previous software [10]. We applied the end-expiratory manoeuver plateau temperature (PLET) method [11]. PLET values depict the exhaled gas temperature within the device and therefore may differ from airway temperature depending on the type of device, respiratory filters and other technological items. For measurements of the temperature in the bronchial lumen and in mucopurulent material within the bronchi, a computerized Clark type oxygen probe (length: 65 cm; outer diameter: 2 mm; inner diameter: 0.4 mm; Licox pO2; GMS, Kiel, Germany) was inserted in the working channel of the bronchoscope and guided under video control into the right upper lobe, obstructed with a mucopurulent material. Additionally, temperature measurements were conducted using a prototype thermistor based probe (Exacon Scientific, Roskilde, Denmark) with a confirmed tolerance of less than ± 0.1 °C between 25° and 45 °C (length: 200 cm; outer diameter: 2 mm; shielded with autoclavable medical grade silicone). The base unit used was a DATEX CARDIOCAP

Please cite this article as: Schmidt A, et al, Neutrophil elastase-mediated increase in airway temperature during inflammation, J Cyst Fibros (2014), http://dx.doi.org/ 10.1016/j.jcf.2014.03.004

A. Schmidt et al. / Journal of Cystic Fibrosis xx (2014) xxx–xxx

II Patient Monitor (Datex Ohmeda, GE Healthcare, Salt Lake City, USA) with a calibrated A type adapter and a preset measuring reference of 2252 Ohms at 25 °C. Patients were mildly sedated. Temperature measurements were made in the airway lumen and in mucopurulent material when present.

2.3. Isothermal titration calorimetry For isothermal titration calorimetry, we incubated purified human NE and α1-PI at ratios of 1:10 or 10:1 w/w assuming extinction coefficients (E) and molecular masses (Mr) for NE of E = 9.85 (1%, 1 cm pathway, 280 nm) and Mr = 30,000 Da [12] and for α1-PI E = 0.433 (0.1%, 1 cm pathway, 280 nm) and Mr = 52,000 Da [13], for 5 min and determined the enthalpy of the reaction mixtures. NE into α1-PI titrations and opposite titrations was performed using an ITC-200 calorimeter (Microcal, Northampton, MA, USA). Titration experiments were performed in the range of 10 °C to 40 °C. Experiments were analysed using the ORIGIN software provided with the calorimeter. A binding model with identical and independent binding sites was used to fit the data.

2.4. Pouch temperature in mouse strains NE-deficient (NE−/−) mice were generated by targeted mutagenesis as previously described [14]. Mouse strains were back-crossed (8 generations) on a pure C57BL6/J background. C57BL/6 NE−/− mice and their wild-type (WT) littermates were used in this study. Mice were sex and age (8–10 weeks) matched and maintained in the animal barrier facility with a 12 h light/ dark cycle and provided with water and food ad libitum. Animal handling and procedures were approved by the Institutional Animal Studies Committee (Health and Animal Protection Office, Châlons-en-Champagne, Authorisation number: 51-31) in accordance with the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA) and following the European Directive 2010/63/EU on the protection of animals used in scientific procedures. Air pouches were generated as previously described [15]. Briefly, 1 ml of sterile air was injected subcutaneously into the back of mice (n = 5 per genotype), followed by instillation of 1 × 10 7 Pseudomonas aeruginosa colony forming units (CFU) per pouch to induce an inflammatory response. We used the P. aeruginosa strain H103, kindly provided by Dr. R. Hancock, Vancouver, BC, Canada, which was grown overnight in Luria Bertani broth at 37 °C to late exponential phase and washed twice with PBS, pH 7.4. Control mice (n = 5) were challenged with sterile PBS alone. At designated time points (three days post-challenge), temperature in the pouch airspace was determined using a temperature probe (C8.B; GMS, Milkendorf, Germany), attached to a Licox CMP temperature monitor (Integra, Plainsboro, NJ, USA) which was introduced into the pouch via an 18 gauge needle. Next, the air pouch was lavaged with PBS, and total and differential counts were performed immediately [16].

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2.5. P. aeruginosa densities at different temperature The following P. aeruginosa strains were used: PAO1 [17], PAO1 SD4 (Δ mucA), and PAO1 SD40 (Δ mucA algT), both DTU Lyngby, Denmark [18]. Strains were grown in trypticase soy broth (TSB) overnight. An aliquot was inoculated in fresh medium to an optical density (OD578) of 0.05, corresponding to colony forming units (CFU) of 1 × 107 to 1 × 108. Then the bacterial suspensions were grown anaerobically in a anaerobic box (Anaerocult®, Merck KGaA, Darmstadt, Germany) using AnaeroGen (Oxoid Ltd., Basingstoke, England) under shaking at 30 °C, 37 °C, 38 °C and 39 °C (Infors, Bottmingen, Switzerland) for 96 h and CFU was determined by plating method. 2.6. P. aeruginosa transcript analysis at different temperatures For P. aeruginosa transcript analysis at different temperatures and aerobic versus anaerobic growth conditions, strain PAO1 was incubated overnight at 30 °C and 38 °C in an 100 ml Erlenmeyer flask, filled with 50 ml TSB medium under shaking at 180 rpm. An aliquot (OD578 0.05) is transferred to a Biostat B plus bioreactor (Sartorius, Melsungen, Germany) containing 1.5 l TSB medium, and incubated at 30 °C and 38 °C aerobically with pH control for 6 h leading to a cell density of 1 × 1010 CFU (OD578 8.0). Then the bioreactor was flushed with nitrogen for 1 h to obtain anaerobic culture conditions. Fifty millilitres of the suspension is harvested and mixed with an equal volume of RNA-Protect buffer (Qiagen, Hilden, Germany). After 10 min incubation at room temperature, 1 ml aliquots are centrifuged at 6000 ×g for 5 min, the supernatant removed and the pellets stored at − 70 °C until transcript analysis. 2.7. Preparation and sequencing of cDNA libraries The preparation and sequencing of the cDNA libraries were done as described previously [19] with some modifications. The libraries were sequenced on an Illumina HiSeq 2000. Barcoded 59-adapters [19] enabled the pooling of multiple samples on one lane of the Illumina flow cell. Libraries were sequenced with 50 cycles in single end mode. Computational analysis was done as described previously [19] with some modifications. The reads were aligned to the PAO1 reference genome using stampy, a short-read aligner that allows for gapped alignments [20]. The reported gene read counts were used to estimate the differential gene expression making use of the package DESeq in R (Project for statistical computing). For details, see [19]. 2.8. LPS analysis For analysis by gel electrophoresis and silver staining, LPS was prepared and visualized as previously described [21]. Monosaccharides (as alditol acetates), total fatty acids (as methyl esters) and organic phosphate contents were determined as reported [21]. GLC analyses were performed either on an Agilent

Please cite this article as: Schmidt A, et al, Neutrophil elastase-mediated increase in airway temperature during inflammation, J Cyst Fibros (2014), http://dx.doi.org/ 10.1016/j.jcf.2014.03.004

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Neutrophil elastase-mediated increase in airway temperature during inflammation.

How elevated temperature is generated during airway infections represents a hitherto unresolved physiological question. We hypothesized that innate im...
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