AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
Page 1 of 41
1
IL-17A is elevated in end-stage COPD and contributes to
2
cigarette smoke-induced lymphoid neogenesis
3 4
Abraham B Roos, PhD1,2, Caroline Sandén, PhD1, Michiko Mori, PhD1, Leif Bjermer, MD,
5
PhD3, Martin R Stampfli, PhD2, 4*, Jonas S Erjefält, PhD1*
6 7
1
Department of Experimental Medical Science, Lund University, Lund, Sweden
8
2
Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre,
9
McMaster University, Hamilton, ON, Canada
10
3
Department of Respiratory Medicine and Allergology, Lund University, Lund, Sweden
11
4
Department of Medicine, Firestone Institute of Respiratory Health at St. Joseph’s Health
12
Care, Hamilton, ON, Canada
13
*These authors contributed equally
14 15
Correspondence and requests for reprints should be addressed to Abraham B Roos,
16
Department of Pathology and Molecular Medicine, McMaster University, MDCL 4084, 1200
17
Main Street West, Hamilton, ON L8K 4P1, Canada. Phone: +1-905-525-9140 ext. 22150. Fax
18
+1-905-522-6750. Email: [email protected].
19 20
Financial Support: Canadian Institutes of Health Research (MOP-64390 and MOP-87517),
21
the Swedish Heart-lung Foundation, and the Swedish Research Council.
22
Short running title: IL-17A in end-stage COPD
23
Descriptor number: COPD
24
Total word count: 3678.
1 Copyright © 2015 by the American Thoracic Society
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
25
Author contributions: Conception and design: ABR, CS, MM, MRS and JSE. Acquisition of
26
data: ABR, CS, MM, LB, and JSE. Analysis and interpretation: ABR, CS, MM, LB, MRS and
27
JSE. Drafting the manuscript for important intellectual content: ABR, CS, JSE and MRS. All
28
authors approved the final version to be published.
29
AT A GLANCE COMMENTARY
30
Scientific Knowledge on the Subject
31
Interleukin (IL)-17A is implicated in mild-severe chronic obstructive pulmonary disease
32
(COPD) but the relation to disease severity and the potential function in end-stage disease are
33
unknown.
34
What This Study Adds to the Field
35
We demonstrate an elevated expression of lung tissue IL-17A in end-stage COPD and provide
36
mechanistic evidence for IL-17A in cigarette smoke-induced lymphoid neogenesis via
37
induction of CXCL12. These findings suggest that IL-17A contributes to the pathology in
38
end-stage COPD.
39 40
This article has an online data supplement, which is accessible from this issue's table of
41
content online at www.atsjournals.org.
2 Copyright © 2015 by the American Thoracic Society
Page 2 of 41
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
Page 3 of 41
42 43 44
ABSTRACT
45
an accumulation of pulmonary lymphoid follicles. Interleukin (IL)-17A is implicated in
46
COPD and pulmonary lymphoid neogenesis in response to microbial stimuli. We
47
hypothesized that IL-17A is increased in peripheral lung tissue during end-stage COPD and
48
also directly contributes to cigarette smoke-induced lymphoid neogenesis.
49
OBJECTIVE: Characterize the tissue expression and functional role of IL-17A in end-stage
50
COPD.
51
METHODS: Automated immune-detection of IL-17A and IL-17F was performed in lung
52
tissue specimens collected from patients with GOLD stage I-IV COPD, as well as smoking
53
and never-smoking controls. In parallel, Il17a-/- mice and WT controls were exposed to
54
cigarette smoke for 24 weeks and pulmonary lymphoid neogenesis was assessed.
55
MEASUREMENTS AND MAIN RESULTS: Tissue expression of IL-17A and IL-17F was
56
increased in COPD and correlated with lung function decline. IL-17A was significantly
57
elevated in severe-very severe COPD (GOLD III/IV), compared to both smokers and never-
58
smokers without COPD. While CD3+ T cells expressed IL-17A in very severe COPD, the
59
majority of IL-17A+ cells were identified as tryptase+ mast cells. Attenuated lymphoid
60
neogenesis and reduced expression of the B cell attracting chemokine C-X-C motif ligand
61
(CXCL)12 was observed in cigarette smoke-exposed Il17a-/- mice. CXCL12 was also highly
62
expressed in lymphoid follicles in COPD lungs, and the pulmonary expression was
63
significantly elevated in end-stage COPD.
64
CONCLUSION: IL-17A in the peripheral lung of patients with severe-very severe COPD
65
may contribute to disease progression and development of lymphoid follicles via activation of
66
CXCL12.
RATIONALE: End-stage chronic obstructive pulmonary disease (COPD) is associated with
3 Copyright © 2015 by the American Thoracic Society
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
67
INTRODUCTION
68
Chronic obstructive pulmonary disease (COPD) is a small airways disease characterized by a
69
progressive and largely irreversible decline in lung function (1). The pathology of COPD is
70
complex and includes airway inflammation and in most patients destruction of the pulmonary
71
parenchyma (emphysema). In addition, formation of tertiary lymphoid tissue is viewed as a
72
characteristic feature of advanced COPD (2, 3). The molecular mechanisms contributing to
73
lymphoid follicle neogenesis in end-stage COPD have, however, not been fully characterized.
74
Interleukin (IL)-17A and IL-17F are pro-inflammatory mediators important for
75
host defenses against microbial and fungal agents (4-6). In addition, critical roles have since
76
the initial discovery been proposed for IL-17A in autoimmune disorders (4, 5) and formation
77
of tertiary lymphoid tissue (7, 8). Transgenic mice prone to the development of lymphoid
78
follicles display an elevated pulmonary IL-17A signature (9). Furthermore, several
79
investigators have demonstrated a requirement for IL-17 signaling in the development of
80
lymphoid follicles induced by bacterial and viral stimuli (7, 8).
81
Previous studies have investigated the lung tissue expression of IL-17A and IL-
82
17F in stable COPD. While the majority of studies reported an increased number of IL-17A+
83
cells in the submucosa of COPD patients (10-12), elevated expression of IL-17F has not been
84
consistently observed (13). Furthermore, the expression and cellular source of IL-17A/F in the
85
distal lung of end-stage COPD remain unknown. Also, the contribution of IL-17A in cigarette
86
smoke (CS)-induced lymphoid neogenesis, a charactersistic feature of end-stage COPD, has
87
not been investigated to date.
88
In this study, we hypothesized that IL-17A/F are increased in peripheral COPD
89
lungs, especially at severe disease stages, and contributes to lymphoid neogenesis that
90
characterizes end stage disease. Our findings demonstrate that end-stage COPD is associated
91
with elevated IL-17A. We furthermore show that IL-17A mechanistically contributes to the
4 Copyright © 2015 by the American Thoracic Society
Page 4 of 41
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
Page 5 of 41
92
development of CS-induced lymphoid follicles by activating the expression of CXCL12.
93
Collectively our data thus identify a novel and potentially important role for IL-17A in COPD
94
pathogeneis.
5 Copyright © 2015 by the American Thoracic Society
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
95
MATERIALS AND METHODS
96
Study cohort and tissue collection
97
Lung tissues were collected at Skåne University Hospital, in Lund, Sweden, as described
98
previously (2). Lung resection samples were obtained from patients with mild-severe COPD
99
(GOLD stage I-III), as well as never smokers and smokers, all undergoing surgery for
100
bronchial tumour. Tissue was also collected from explanted lungs from patients with very
101
severe (GOLD IV) COPD, without a history of lung cancer. All COPD patients (n=30) had
102
stable disease. The GOLD criteria (14) was used to classify patients to mild-moderate (GOLD
103
stage I-II, n=18) and severe-very severe (GOLD stage III-IV, n=12) COPD. Never-smokers
104
and ex- or current smokers with normal lung function were also included (n = 15). Clinical
105
characteristics are described in Table 1. Randomly selected lobes were immediately immersed
106
in 4% paraformaldehyde, and embedded in paraffin. The study was approved by the local
107
Swedish Research Ethics Committee in Lund, Sweden. All patients signed informed consent
108
to participate.
109 110
Immunohistochemical assessment
111
Immunostaining was performed on 4 µm sections using an automated slide stainer
112
(Autostainer Plus, DakoCytomation, Glostrup, Denmark). Antibodies and details are
113
presented in Table 2. Slides were counterstained with DAPI or Mayer’s haematoxylin.
114
Specificity was confirmed by omitting the primary antibody, and a subsequent lack of
115
staining. All immunodetection was performed simultaneously to avoid variability in staining
116
intensity. Immunohistochemical staining was performed on 1-3 sections/subject.
117
High-resolution digital images were generated using a ScanScope Slide Scanner
118
(Aperio Technologies, Vista, CA, USA). Morphometric analyses were accomplished using
119
Aperio ImageScope v.10.0 software (Aperio Technologies), as previously described (15). A
6 Copyright © 2015 by the American Thoracic Society
Page 6 of 41
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
Page 7 of 41
120
pixel threshold for positivity was set to include positive stained pixels using the Aperio
121
Positive Pixel Count Algorithm v.9 (Aperio Technologies). The immunoreactivity was
122
calculated by an investigator blinded to the protocol.
123 124
Animals
125
6-8 week old female C57BL/6 mice were supplied by Jackson Laboratories (Bar Harbor, ME,
126
USA). Il17a-/- mice (C57BL/6 background) (generated by Dr. Yoichiro Iwakura, University of
127
Tokyo, Tokyo (16), kindly provided by Dr. Jay Kolls, University of Pittsburgh, PA) were
128
bred in-house. Food and water was provided ad libitum. All mice were housed with a light-
129
dark cycle of 12 hours, under specific pathogen-free conditions. Experiments were approved
130
by the Animal Research Ethics Board at McMaster University and conducted in accordance
131
with the ethical guidelines outlined by the Canadian Council on Animal Care.
132
Animals were exposed to 12 3R4F reference cigarettes (Tobacco and Health
133
Research Institute, University of Kentucky, Lexington, KY, USA) with the filters removed in
134
a SIU-48 whole body cigarette smoking machine (Promech Lab AB, Vintrie, Sweden) (30).
135
Mice were exposed two times daily for 50 minutes, five days a week for 24 weeks
136
consecutive days. Control mice were exposed to room air only.
137
Lungs were insufflated with 10% formalin under constant pressure. The number
138
of B cell (B220+) aggregates with a dense B cell core and >50 cells (defined as lymphoid
139
follicles), as well as B cell aggregates with fewer cells and/or dispersed B cell localization
140
(defined as B cell clusters) were enumerated along with the number of CXCL12 positive cells
141
in lymphoid follicles/aggregates or surrounding airways. Long term cigarette smoke exposure
142
was performed once.
143
144
In situ hybridization
7 Copyright © 2015 by the American Thoracic Society
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
145
RNAscope 2.0 FFPE assay kit was used to perform in situ hybridization for Il17a RNA on 4
146
µm tissue sections, according to the manufacturer’s instructions (Advanced Cell Diagnostics,
147
CA, USA). Deparaffinized tissue was treated with heat and protease, and hybridized to the
148
target probe. Amplified target RNA was detected using RNAscope 2.0 HD brown reagent kit
149
(Advanced Cell Diagnostics) and counterstained with Gill’s Hematoxylin. Probes targeting
150
PPIB or the bacterial gene DapB were used as positive/negative controls. Digital images were
151
generated with a ScanScope Slide Scanner (Aperio Technologies).
152 153
Statistical analysis
154
All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc, La
155
Jolla, CA). One-way ANOVA with Dunnet’s multiple test comparison was used to detect
156
significant differences between never-smoking/smoking controls and GOLD I-IV COPD.
157
Differences were considered statistically significant at p
Page 1 of 41
1
IL-17A is elevated in end-stage COPD and contributes to
2
cigarette smoke-induced lymphoid neogenesis
3 4
Abraham B Roos, PhD1,2, Caroline Sandén, PhD1, Michiko Mori, PhD1, Leif Bjermer, MD,
5
PhD3, Martin R Stampfli, PhD2, 4*, Jonas S Erjefält, PhD1*
6 7
1
Department of Experimental Medical Science, Lund University, Lund, Sweden
8
2
Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre,
9
McMaster University, Hamilton, ON, Canada
10
3
Department of Respiratory Medicine and Allergology, Lund University, Lund, Sweden
11
4
Department of Medicine, Firestone Institute of Respiratory Health at St. Joseph’s Health
12
Care, Hamilton, ON, Canada
13
*These authors contributed equally
14 15
Correspondence and requests for reprints should be addressed to Abraham B Roos,
16
Department of Pathology and Molecular Medicine, McMaster University, MDCL 4084, 1200
17
Main Street West, Hamilton, ON L8K 4P1, Canada. Phone: +1-905-525-9140 ext. 22150. Fax
18
+1-905-522-6750. Email: [email protected].
19 20
Financial Support: Canadian Institutes of Health Research (MOP-64390 and MOP-87517),
21
the Swedish Heart-lung Foundation, and the Swedish Research Council.
22
Short running title: IL-17A in end-stage COPD
23
Descriptor number: COPD
24
Total word count: 3678.
1 Copyright © 2015 by the American Thoracic Society
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
25
Author contributions: Conception and design: ABR, CS, MM, MRS and JSE. Acquisition of
26
data: ABR, CS, MM, LB, and JSE. Analysis and interpretation: ABR, CS, MM, LB, MRS and
27
JSE. Drafting the manuscript for important intellectual content: ABR, CS, JSE and MRS. All
28
authors approved the final version to be published.
29
AT A GLANCE COMMENTARY
30
Scientific Knowledge on the Subject
31
Interleukin (IL)-17A is implicated in mild-severe chronic obstructive pulmonary disease
32
(COPD) but the relation to disease severity and the potential function in end-stage disease are
33
unknown.
34
What This Study Adds to the Field
35
We demonstrate an elevated expression of lung tissue IL-17A in end-stage COPD and provide
36
mechanistic evidence for IL-17A in cigarette smoke-induced lymphoid neogenesis via
37
induction of CXCL12. These findings suggest that IL-17A contributes to the pathology in
38
end-stage COPD.
39 40
This article has an online data supplement, which is accessible from this issue's table of
41
content online at www.atsjournals.org.
2 Copyright © 2015 by the American Thoracic Society
Page 2 of 41
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
Page 3 of 41
42 43 44
ABSTRACT
45
an accumulation of pulmonary lymphoid follicles. Interleukin (IL)-17A is implicated in
46
COPD and pulmonary lymphoid neogenesis in response to microbial stimuli. We
47
hypothesized that IL-17A is increased in peripheral lung tissue during end-stage COPD and
48
also directly contributes to cigarette smoke-induced lymphoid neogenesis.
49
OBJECTIVE: Characterize the tissue expression and functional role of IL-17A in end-stage
50
COPD.
51
METHODS: Automated immune-detection of IL-17A and IL-17F was performed in lung
52
tissue specimens collected from patients with GOLD stage I-IV COPD, as well as smoking
53
and never-smoking controls. In parallel, Il17a-/- mice and WT controls were exposed to
54
cigarette smoke for 24 weeks and pulmonary lymphoid neogenesis was assessed.
55
MEASUREMENTS AND MAIN RESULTS: Tissue expression of IL-17A and IL-17F was
56
increased in COPD and correlated with lung function decline. IL-17A was significantly
57
elevated in severe-very severe COPD (GOLD III/IV), compared to both smokers and never-
58
smokers without COPD. While CD3+ T cells expressed IL-17A in very severe COPD, the
59
majority of IL-17A+ cells were identified as tryptase+ mast cells. Attenuated lymphoid
60
neogenesis and reduced expression of the B cell attracting chemokine C-X-C motif ligand
61
(CXCL)12 was observed in cigarette smoke-exposed Il17a-/- mice. CXCL12 was also highly
62
expressed in lymphoid follicles in COPD lungs, and the pulmonary expression was
63
significantly elevated in end-stage COPD.
64
CONCLUSION: IL-17A in the peripheral lung of patients with severe-very severe COPD
65
may contribute to disease progression and development of lymphoid follicles via activation of
66
CXCL12.
RATIONALE: End-stage chronic obstructive pulmonary disease (COPD) is associated with
3 Copyright © 2015 by the American Thoracic Society
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
67
INTRODUCTION
68
Chronic obstructive pulmonary disease (COPD) is a small airways disease characterized by a
69
progressive and largely irreversible decline in lung function (1). The pathology of COPD is
70
complex and includes airway inflammation and in most patients destruction of the pulmonary
71
parenchyma (emphysema). In addition, formation of tertiary lymphoid tissue is viewed as a
72
characteristic feature of advanced COPD (2, 3). The molecular mechanisms contributing to
73
lymphoid follicle neogenesis in end-stage COPD have, however, not been fully characterized.
74
Interleukin (IL)-17A and IL-17F are pro-inflammatory mediators important for
75
host defenses against microbial and fungal agents (4-6). In addition, critical roles have since
76
the initial discovery been proposed for IL-17A in autoimmune disorders (4, 5) and formation
77
of tertiary lymphoid tissue (7, 8). Transgenic mice prone to the development of lymphoid
78
follicles display an elevated pulmonary IL-17A signature (9). Furthermore, several
79
investigators have demonstrated a requirement for IL-17 signaling in the development of
80
lymphoid follicles induced by bacterial and viral stimuli (7, 8).
81
Previous studies have investigated the lung tissue expression of IL-17A and IL-
82
17F in stable COPD. While the majority of studies reported an increased number of IL-17A+
83
cells in the submucosa of COPD patients (10-12), elevated expression of IL-17F has not been
84
consistently observed (13). Furthermore, the expression and cellular source of IL-17A/F in the
85
distal lung of end-stage COPD remain unknown. Also, the contribution of IL-17A in cigarette
86
smoke (CS)-induced lymphoid neogenesis, a charactersistic feature of end-stage COPD, has
87
not been investigated to date.
88
In this study, we hypothesized that IL-17A/F are increased in peripheral COPD
89
lungs, especially at severe disease stages, and contributes to lymphoid neogenesis that
90
characterizes end stage disease. Our findings demonstrate that end-stage COPD is associated
91
with elevated IL-17A. We furthermore show that IL-17A mechanistically contributes to the
4 Copyright © 2015 by the American Thoracic Society
Page 4 of 41
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
Page 5 of 41
92
development of CS-induced lymphoid follicles by activating the expression of CXCL12.
93
Collectively our data thus identify a novel and potentially important role for IL-17A in COPD
94
pathogeneis.
5 Copyright © 2015 by the American Thoracic Society
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
95
MATERIALS AND METHODS
96
Study cohort and tissue collection
97
Lung tissues were collected at Skåne University Hospital, in Lund, Sweden, as described
98
previously (2). Lung resection samples were obtained from patients with mild-severe COPD
99
(GOLD stage I-III), as well as never smokers and smokers, all undergoing surgery for
100
bronchial tumour. Tissue was also collected from explanted lungs from patients with very
101
severe (GOLD IV) COPD, without a history of lung cancer. All COPD patients (n=30) had
102
stable disease. The GOLD criteria (14) was used to classify patients to mild-moderate (GOLD
103
stage I-II, n=18) and severe-very severe (GOLD stage III-IV, n=12) COPD. Never-smokers
104
and ex- or current smokers with normal lung function were also included (n = 15). Clinical
105
characteristics are described in Table 1. Randomly selected lobes were immediately immersed
106
in 4% paraformaldehyde, and embedded in paraffin. The study was approved by the local
107
Swedish Research Ethics Committee in Lund, Sweden. All patients signed informed consent
108
to participate.
109 110
Immunohistochemical assessment
111
Immunostaining was performed on 4 µm sections using an automated slide stainer
112
(Autostainer Plus, DakoCytomation, Glostrup, Denmark). Antibodies and details are
113
presented in Table 2. Slides were counterstained with DAPI or Mayer’s haematoxylin.
114
Specificity was confirmed by omitting the primary antibody, and a subsequent lack of
115
staining. All immunodetection was performed simultaneously to avoid variability in staining
116
intensity. Immunohistochemical staining was performed on 1-3 sections/subject.
117
High-resolution digital images were generated using a ScanScope Slide Scanner
118
(Aperio Technologies, Vista, CA, USA). Morphometric analyses were accomplished using
119
Aperio ImageScope v.10.0 software (Aperio Technologies), as previously described (15). A
6 Copyright © 2015 by the American Thoracic Society
Page 6 of 41
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
Page 7 of 41
120
pixel threshold for positivity was set to include positive stained pixels using the Aperio
121
Positive Pixel Count Algorithm v.9 (Aperio Technologies). The immunoreactivity was
122
calculated by an investigator blinded to the protocol.
123 124
Animals
125
6-8 week old female C57BL/6 mice were supplied by Jackson Laboratories (Bar Harbor, ME,
126
USA). Il17a-/- mice (C57BL/6 background) (generated by Dr. Yoichiro Iwakura, University of
127
Tokyo, Tokyo (16), kindly provided by Dr. Jay Kolls, University of Pittsburgh, PA) were
128
bred in-house. Food and water was provided ad libitum. All mice were housed with a light-
129
dark cycle of 12 hours, under specific pathogen-free conditions. Experiments were approved
130
by the Animal Research Ethics Board at McMaster University and conducted in accordance
131
with the ethical guidelines outlined by the Canadian Council on Animal Care.
132
Animals were exposed to 12 3R4F reference cigarettes (Tobacco and Health
133
Research Institute, University of Kentucky, Lexington, KY, USA) with the filters removed in
134
a SIU-48 whole body cigarette smoking machine (Promech Lab AB, Vintrie, Sweden) (30).
135
Mice were exposed two times daily for 50 minutes, five days a week for 24 weeks
136
consecutive days. Control mice were exposed to room air only.
137
Lungs were insufflated with 10% formalin under constant pressure. The number
138
of B cell (B220+) aggregates with a dense B cell core and >50 cells (defined as lymphoid
139
follicles), as well as B cell aggregates with fewer cells and/or dispersed B cell localization
140
(defined as B cell clusters) were enumerated along with the number of CXCL12 positive cells
141
in lymphoid follicles/aggregates or surrounding airways. Long term cigarette smoke exposure
142
was performed once.
143
144
In situ hybridization
7 Copyright © 2015 by the American Thoracic Society
AJRCCM Articles in Press. Published on 06-April-2015 as 10.1164/rccm.201410-1861OC
145
RNAscope 2.0 FFPE assay kit was used to perform in situ hybridization for Il17a RNA on 4
146
µm tissue sections, according to the manufacturer’s instructions (Advanced Cell Diagnostics,
147
CA, USA). Deparaffinized tissue was treated with heat and protease, and hybridized to the
148
target probe. Amplified target RNA was detected using RNAscope 2.0 HD brown reagent kit
149
(Advanced Cell Diagnostics) and counterstained with Gill’s Hematoxylin. Probes targeting
150
PPIB or the bacterial gene DapB were used as positive/negative controls. Digital images were
151
generated with a ScanScope Slide Scanner (Aperio Technologies).
152 153
Statistical analysis
154
All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc, La
155
Jolla, CA). One-way ANOVA with Dunnet’s multiple test comparison was used to detect
156
significant differences between never-smoking/smoking controls and GOLD I-IV COPD.
157
Differences were considered statistically significant at p
