Accepted Article 1
Nitric Oxide Production by Polymorphonuclear Leukocytes in Infected Cystic Fibrosis
2
Sputum Consumes Oxygen 1
3 4
Mette Kolpen1,2, Thomas Bjarnsholt1,2, Claus Moser1, Christine R. Hansen3, Lars F. Rickelt4,
5
Michael Kühl4,5,6, Casper Hempel1,2, , Tanja Pressler3, Niels Høiby1,2, Peter Ø. Jensen1.
6 7
1
Department of Clinical Microbiology, Rigshospitalet, Copenhagen
8
2
Department of International Health, Immunology and Microbiology, Faculty of Health Sciences
9
University of Copenhagen, Denmark
10
3
Copenhagen CF Centre, Rigshospitalet, Copenhagen, Denmark
11
4
Marine Biological Laboratory, Department of Biology, University of Copenhagen, Denmark
12
5
Plant Functional Biology and Climate Change Cluster, University of Technology Sydney,
13
Australia
14
6
15
University, Singapore
Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological
16 17
Correspondence: Peter Østrup Jensen, Department of Clinical Microbiology, Rigshospitalet, Ole
18
Maaloesvej 26, 2100 Copenhagen, Denmark, Tel: + 45 35 45 78 08, E-mail:
19
[email protected]
20 21 22
Running title: NO production by PMNs in infected CF sputum
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cei.12318
1 This article is protected by copyright. All rights reserved.
Accepted Article 23
24
Summary
25
Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by
26
persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by
27
numerous polymorphonuclear leukocytes (PMNs), which consume a major part of present
28
molecular oxygen (O2) due to production of superoxide (O2-). In this study, we show that the PMNs
29
also consume O2 for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the
30
infected endobronchial. Fresh expectorated sputum samples (n = 28) from chronically infected CF
31
patients (n = 22) were analyzed by quantifying and visualizing the NO production. NO production
32
was detected by optode measurements combined with fluorescence microscopy, flow cytometry and
33
spectrophotometry. Inhibition of nitric oxide synthases (NOS) with NG-monomethyl-L-arginine (L-
34
NMMA) resulted in reduced O2 consumption (p2 precipitating antibodies,
103
normal: 0–1) [19]. The experiments were performed before elective intravenous antibiotics
104
treatment of the CF patients for 2 weeks every 3-4 months [20;21].
105 106
Concentration of dissolved O2. Sputum samples were equilibrated to normoxic conditions by
107
diluting 10 times with Krebs–Ringer buffer (130 mM NaCl, 5 mM KCl, 0.9 mM CaCl2, 1.2 mM
108
MgCl2, 15 mM NaH2PO4 (pH = 7.4)) with 10 mM glucose equilibrated in ambient air. Dissolved O2
109
in the sample was measured in a reaction chamber fixed on top of a luminescent dissolved oxygen
110
(LDO) sensor connected to an HQ40d meter (HACH Company, Loveland, CO, USA) as previously
111
described [5]. To separate the O2 consumed by NOS from total consumption, sputum was pretreated
112
with the NOS-inhibitor, 2 mM NG-monomethyl-L-arginine (L-NMMA) (M7033, Sigma, St Louis,
113
Missouri, USA), for 10 min at 37 oC before recording of the concentration of O2 in the samples for
114
30 min.
115
5 This article is protected by copyright. All rights reserved.
Accepted Article 116
Visualization of NO production: Sputum was stained with 20 M 4-amino-5-methylamino-2',7'-
117
difluorofluorescein diacetate (DAF-FM) (D23842, Life Technologies, Copenhagen,DK) and shaken
118
for 30 min at 37oC. To verify that the NO content was related to NOS, sputum samples were
119
pretreated with 2 mM L-NMMA for 10 min before addition of DAF-FM. Detection of PMNs
120
engaged in NO production in sputum was done by staining with 5 M DAF-FM and with 5 M
121
hydroethidine (HE) (D-23107, Life Technologies) to identify PMNs with an ongoing production O2-
122
. After incubation the samples were examined with a fluorescence microscope (BX40, Olympus,
123
Japan) equipped with a FITC-PI filter.
124 125
Quantification of intracellular NO: DAF-FM stained sputum was washed in Krebs-Ringer buffer
126
and fixated in 2% paraformaldehyde (PFA) (P-6148, Sigma-Aldrich, St Louis, MO, US) in
127
phosphate buffered saline (PBS), pH 7.4. Cells in the fixed sputum were stained with propidium
128
iodide (PI) (100 g ml-1) (P-4170, Sigma-Aldrich) before flow cytometry. To avoid influence of the
129
time period between the fixation and flow cytometry, fixated samples were analyzed 15 minutes
130
after fixation.
131 132
Intracellular staining of iNOS in sputum: Sputum was fixated in 2 % PFA in PBS (126101, USB
133
Corporation, Cleveland, OH, US). Fixed cells were permeabilized with 0.2 % Triton X-100
134
(45H04811, Sigma, St Louis, Missouri, US) and washed in PBS before incubation with IgG2a
135
fluorescein isothiocyanate (FITC)-labeled anti-iNOS (BD 610331, Becton Dickinson, Denmark)
136
and the FITC-labeled mouse isotype control (BD 340459, Becton Dickinson) diluted in mouse sera
137
(X0910, DAKO, Denmark) overnight on ice. Cells were stained with PI before flow cytometry.
138
6 This article is protected by copyright. All rights reserved.
Accepted Article 139
PMN concentration: The concentration of PMNs was estimated as previously described [5].
140
Briefly, the concentration of leukocytes in the sputum was determined by adding 100 l diluted
141
sputum to a TrueCount tube (BD 340334, Becton Dickinson) with 400 l Facslysis solution (BD
142
349202, Becton Dickinson) with PI (100 g ml-1) and the samples were incubated for at least 10
143
min prior to flow cytometry. The frequency of PMNs was estimated by addition of 5 l of each of
144
the following mouse anti-human monoclonal antibodies from BD Bioscience: phycoerythrin-
145
labeled CD11b (555388), peridinin chlorophyll A proteinlabelled CD14 (345786), FITC-labelled
146
CD15 (555401), and allophycocyanin-labeled CD45 (555485) to 100 l diluted sputum. The
147
samples were incubated on ice for 30 min, washed with cold PBS, and fixated in PBS with 2% PFA
148
before they were analyzed by flow cytometry. The concentration of PMNs was calculated by
149
multiplying the concentration of leukocytes with the fraction of PMNs.
150 151
Flow cytometry: The samples were analyzed using a FACSort (BD Bioscience, La Jolla, CA, US)
152
equipped with a 15 mV argon-ion laser tuned at 488 nm, and a red diode laser emitting at 635 nm
153
for excitation. Light scatter, time, and exponentially amplified fluorescence parameters from at least
154
10 000 events, were recorded in list mode. Host cells were discriminated according to their DNA
155
content and their morphology by gating on the PI fluorescence intensity and light scatter.
156
Leukocytes were discriminated by gating on CD45 and the PMNs were discriminated by gating on
157
CD11b, CD14 and CD15 as previously described [5]. The instrument was calibrated using Calibrite
158
beads (BD Bioscience).
159 160
NO3- and NO2- quantification: The concentration of NO3- and NO2- in sputum was measured in 11
161
samples using the Griess colorimetric reaction (no. 780001, Cayman Chemicals, USA) according to
162
the manufacturer’s recommendations. From each sputum sample, 100 µL were immediately diluted
7 This article is protected by copyright. All rights reserved.
Accepted Article 163
10x in PBS and stored at -20oC for later analysis. Frozen sputum samples were transferred to a 96
164
well microtiter plate. NO2- concentration was estimated by addition of the Griess Reagent for 10
165
minutes, whereby NO2- was converted into a purple azo-compound, which was quantitated by the
166
optical density at 540-550 nm measured with an ELISA plate reader (Thermo Scientific Multiskan
167
EX, Thermo Fisher Scientific Inc, BioImage, Denmark). Total NO3- and NO2- levels were estimated
168
by a two-step analysis process: The first step converted NO3- to NO2- utilizing NO3- reductase. After
169
incubation for 2 hours, the next step involved the addition of the Griess Reagent, whereby NO2- was
170
converted into a purple azo-compound. After incubation with Griess Reagent for 10 minutes, the
171
optical density at 540-550 nm was measured with an ELISA plate reader (Thermo Scientific
172
Multiskan EX, Thermo Fisher Scientific Inc, BioImage, Denmark). A NO3- standard curve was used
173
for determination of total NO3- and NO2- concentration, while a NO2- standard curve was used for
174
determination of NO2- alone. The concentration of NO3- was calculated as the difference between
175
the NO3- concentration and the total NO3- and NO2- concentration.
176 177
NO-microprofile: A sputum sample was added a well in a microtiter plate (NuncTM). A NO-
178
microprofile in the sputum sample was then monitored with an amperometric microsensor
179
(Unisense, Århus, Denmark). The microsensor was prepared and calibrated as previously described
180
[22]. The microsensor tip with a tip diameter of 80 m was adjusted manually to the upper surface
181
of the sample and vertical concentration profiles were measured by using a semiautomated setup.
182
The microsensor was mounted on a micromanipulator (Translation Stage VT-80, MICOS, USA)
183
and connected to a picoammeter PA2000 (Unisense). Measurements were controlled by Sensortrace
184
Pro 2.0 (Unisense). Profile measurements were permitted by movement of the sensor in 100-μm
185
steps through the sputum sample.
186
8 This article is protected by copyright. All rights reserved.
Accepted Article 187 188
Statistical methods:
189
Statistical significance was evaluated by Wilcoxon Signed-Rank test and Spearman Rank
190
Correlation test. A p value
Nitric Oxide Production by Polymorphonuclear Leukocytes in Infected Cystic Fibrosis
2
Sputum Consumes Oxygen 1
3 4
Mette Kolpen1,2, Thomas Bjarnsholt1,2, Claus Moser1, Christine R. Hansen3, Lars F. Rickelt4,
5
Michael Kühl4,5,6, Casper Hempel1,2, , Tanja Pressler3, Niels Høiby1,2, Peter Ø. Jensen1.
6 7
1
Department of Clinical Microbiology, Rigshospitalet, Copenhagen
8
2
Department of International Health, Immunology and Microbiology, Faculty of Health Sciences
9
University of Copenhagen, Denmark
10
3
Copenhagen CF Centre, Rigshospitalet, Copenhagen, Denmark
11
4
Marine Biological Laboratory, Department of Biology, University of Copenhagen, Denmark
12
5
Plant Functional Biology and Climate Change Cluster, University of Technology Sydney,
13
Australia
14
6
15
University, Singapore
Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological
16 17
Correspondence: Peter Østrup Jensen, Department of Clinical Microbiology, Rigshospitalet, Ole
18
Maaloesvej 26, 2100 Copenhagen, Denmark, Tel: + 45 35 45 78 08, E-mail:
19
[email protected]
20 21 22
Running title: NO production by PMNs in infected CF sputum
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cei.12318
1 This article is protected by copyright. All rights reserved.
Accepted Article 23
24
Summary
25
Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by
26
persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by
27
numerous polymorphonuclear leukocytes (PMNs), which consume a major part of present
28
molecular oxygen (O2) due to production of superoxide (O2-). In this study, we show that the PMNs
29
also consume O2 for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the
30
infected endobronchial. Fresh expectorated sputum samples (n = 28) from chronically infected CF
31
patients (n = 22) were analyzed by quantifying and visualizing the NO production. NO production
32
was detected by optode measurements combined with fluorescence microscopy, flow cytometry and
33
spectrophotometry. Inhibition of nitric oxide synthases (NOS) with NG-monomethyl-L-arginine (L-
34
NMMA) resulted in reduced O2 consumption (p2 precipitating antibodies,
103
normal: 0–1) [19]. The experiments were performed before elective intravenous antibiotics
104
treatment of the CF patients for 2 weeks every 3-4 months [20;21].
105 106
Concentration of dissolved O2. Sputum samples were equilibrated to normoxic conditions by
107
diluting 10 times with Krebs–Ringer buffer (130 mM NaCl, 5 mM KCl, 0.9 mM CaCl2, 1.2 mM
108
MgCl2, 15 mM NaH2PO4 (pH = 7.4)) with 10 mM glucose equilibrated in ambient air. Dissolved O2
109
in the sample was measured in a reaction chamber fixed on top of a luminescent dissolved oxygen
110
(LDO) sensor connected to an HQ40d meter (HACH Company, Loveland, CO, USA) as previously
111
described [5]. To separate the O2 consumed by NOS from total consumption, sputum was pretreated
112
with the NOS-inhibitor, 2 mM NG-monomethyl-L-arginine (L-NMMA) (M7033, Sigma, St Louis,
113
Missouri, USA), for 10 min at 37 oC before recording of the concentration of O2 in the samples for
114
30 min.
115
5 This article is protected by copyright. All rights reserved.
Accepted Article 116
Visualization of NO production: Sputum was stained with 20 M 4-amino-5-methylamino-2',7'-
117
difluorofluorescein diacetate (DAF-FM) (D23842, Life Technologies, Copenhagen,DK) and shaken
118
for 30 min at 37oC. To verify that the NO content was related to NOS, sputum samples were
119
pretreated with 2 mM L-NMMA for 10 min before addition of DAF-FM. Detection of PMNs
120
engaged in NO production in sputum was done by staining with 5 M DAF-FM and with 5 M
121
hydroethidine (HE) (D-23107, Life Technologies) to identify PMNs with an ongoing production O2-
122
. After incubation the samples were examined with a fluorescence microscope (BX40, Olympus,
123
Japan) equipped with a FITC-PI filter.
124 125
Quantification of intracellular NO: DAF-FM stained sputum was washed in Krebs-Ringer buffer
126
and fixated in 2% paraformaldehyde (PFA) (P-6148, Sigma-Aldrich, St Louis, MO, US) in
127
phosphate buffered saline (PBS), pH 7.4. Cells in the fixed sputum were stained with propidium
128
iodide (PI) (100 g ml-1) (P-4170, Sigma-Aldrich) before flow cytometry. To avoid influence of the
129
time period between the fixation and flow cytometry, fixated samples were analyzed 15 minutes
130
after fixation.
131 132
Intracellular staining of iNOS in sputum: Sputum was fixated in 2 % PFA in PBS (126101, USB
133
Corporation, Cleveland, OH, US). Fixed cells were permeabilized with 0.2 % Triton X-100
134
(45H04811, Sigma, St Louis, Missouri, US) and washed in PBS before incubation with IgG2a
135
fluorescein isothiocyanate (FITC)-labeled anti-iNOS (BD 610331, Becton Dickinson, Denmark)
136
and the FITC-labeled mouse isotype control (BD 340459, Becton Dickinson) diluted in mouse sera
137
(X0910, DAKO, Denmark) overnight on ice. Cells were stained with PI before flow cytometry.
138
6 This article is protected by copyright. All rights reserved.
Accepted Article 139
PMN concentration: The concentration of PMNs was estimated as previously described [5].
140
Briefly, the concentration of leukocytes in the sputum was determined by adding 100 l diluted
141
sputum to a TrueCount tube (BD 340334, Becton Dickinson) with 400 l Facslysis solution (BD
142
349202, Becton Dickinson) with PI (100 g ml-1) and the samples were incubated for at least 10
143
min prior to flow cytometry. The frequency of PMNs was estimated by addition of 5 l of each of
144
the following mouse anti-human monoclonal antibodies from BD Bioscience: phycoerythrin-
145
labeled CD11b (555388), peridinin chlorophyll A proteinlabelled CD14 (345786), FITC-labelled
146
CD15 (555401), and allophycocyanin-labeled CD45 (555485) to 100 l diluted sputum. The
147
samples were incubated on ice for 30 min, washed with cold PBS, and fixated in PBS with 2% PFA
148
before they were analyzed by flow cytometry. The concentration of PMNs was calculated by
149
multiplying the concentration of leukocytes with the fraction of PMNs.
150 151
Flow cytometry: The samples were analyzed using a FACSort (BD Bioscience, La Jolla, CA, US)
152
equipped with a 15 mV argon-ion laser tuned at 488 nm, and a red diode laser emitting at 635 nm
153
for excitation. Light scatter, time, and exponentially amplified fluorescence parameters from at least
154
10 000 events, were recorded in list mode. Host cells were discriminated according to their DNA
155
content and their morphology by gating on the PI fluorescence intensity and light scatter.
156
Leukocytes were discriminated by gating on CD45 and the PMNs were discriminated by gating on
157
CD11b, CD14 and CD15 as previously described [5]. The instrument was calibrated using Calibrite
158
beads (BD Bioscience).
159 160
NO3- and NO2- quantification: The concentration of NO3- and NO2- in sputum was measured in 11
161
samples using the Griess colorimetric reaction (no. 780001, Cayman Chemicals, USA) according to
162
the manufacturer’s recommendations. From each sputum sample, 100 µL were immediately diluted
7 This article is protected by copyright. All rights reserved.
Accepted Article 163
10x in PBS and stored at -20oC for later analysis. Frozen sputum samples were transferred to a 96
164
well microtiter plate. NO2- concentration was estimated by addition of the Griess Reagent for 10
165
minutes, whereby NO2- was converted into a purple azo-compound, which was quantitated by the
166
optical density at 540-550 nm measured with an ELISA plate reader (Thermo Scientific Multiskan
167
EX, Thermo Fisher Scientific Inc, BioImage, Denmark). Total NO3- and NO2- levels were estimated
168
by a two-step analysis process: The first step converted NO3- to NO2- utilizing NO3- reductase. After
169
incubation for 2 hours, the next step involved the addition of the Griess Reagent, whereby NO2- was
170
converted into a purple azo-compound. After incubation with Griess Reagent for 10 minutes, the
171
optical density at 540-550 nm was measured with an ELISA plate reader (Thermo Scientific
172
Multiskan EX, Thermo Fisher Scientific Inc, BioImage, Denmark). A NO3- standard curve was used
173
for determination of total NO3- and NO2- concentration, while a NO2- standard curve was used for
174
determination of NO2- alone. The concentration of NO3- was calculated as the difference between
175
the NO3- concentration and the total NO3- and NO2- concentration.
176 177
NO-microprofile: A sputum sample was added a well in a microtiter plate (NuncTM). A NO-
178
microprofile in the sputum sample was then monitored with an amperometric microsensor
179
(Unisense, Århus, Denmark). The microsensor was prepared and calibrated as previously described
180
[22]. The microsensor tip with a tip diameter of 80 m was adjusted manually to the upper surface
181
of the sample and vertical concentration profiles were measured by using a semiautomated setup.
182
The microsensor was mounted on a micromanipulator (Translation Stage VT-80, MICOS, USA)
183
and connected to a picoammeter PA2000 (Unisense). Measurements were controlled by Sensortrace
184
Pro 2.0 (Unisense). Profile measurements were permitted by movement of the sensor in 100-μm
185
steps through the sputum sample.
186
8 This article is protected by copyright. All rights reserved.
Accepted Article 187 188
Statistical methods:
189
Statistical significance was evaluated by Wilcoxon Signed-Rank test and Spearman Rank
190
Correlation test. A p value
