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JAK/STAT pathway activation in COPD

1Liang Yew-Booth, 1Mark A. Birrell, 1Ming Sum Lau, 1Katie Baker, 1Victoria Jones, 2Iain Kilty and 1Maria G. Belvisi

1Respiratory Pharmacology, National Heart and Lung Institute, Faculty of Medicine, Imperial

College London, Exhibition Road, London, SW7 2AZ, UK. 2Pfizer Inc, 610 Main Street,

Cambridge MA 02139, USA

Correspondence: Prof Maria Belvisi, Tel: +44 (0)207 594 7828

Fax: +44 (0)207 594 3100, E-mail: [email protected]

Take home message: A comprehensive investigation into the activation status of the JAK-

STATs pathways in the lungs of COPD patients.

Sources of support: Post doctorial funding for L Y-B from Pfizer

Word count: 2553

Word Count: 2337

Abstract

The Janus Kinase/ Signal Transducers and Activators of Transcription (JAK/STAT)

pathways represent critical nodes in inflammatory signalling and inhibitors of these pathways

may represent treatments for chronic obstructive pulmonary disease (COPD). There is,

however, limited target validation data demonstrating an increase in the activation status of

the various JAK/STAT proteins in tissue from COPD patients. Our aim was to determine the

extent of JAK/STAT pathway activation in diseased lung tissue.

We determined the expression of phosphorylated STAT proteins as a marker of JAK/STAT

pathway activation in human parenchymal samples from non-smokers, smokers and COPD

patients by western blotting. Also, to determine the localisation of STAT phosphorylation,

Immunohistochemical (IHC) staining of phospho-STAT proteins was assessed in epithelial

cells, macrophages and the overall parenchymal section.

Western blotting revealed a significant increase in the phosphorylation of tyrosine, but not

serine sites, on STAT1 and 3 in lungs from COPD patients compared to non-smokers.

Analysis of the IHC staining revealed a statistically significant increase in phospho-STAT2 in

the parenchymal macrophages of smokers compared to non-smokers.

These data suggest that the JAK/STAT pathway is activated in COPD patients and provides

encouraging target validation data which supports drug discovery efforts in this area.

1

Introduction

Chronic obstructive pulmonary disease (COPD) is an inflammatory disease of the lung, most

commonly resulting from cigarette smoke (CS) exposure, characterised by a largely

irreversible and progressive airflow limitation [1, 2]. Currently there are no disease-

modifying therapies to prevent the relentless disease progression in patients with COPD. As a

consequence, COPD is associated with high mortality and increasing prevalence worldwide

[2]. Therefore, significant research effort has been directed to understanding the mechanism

by which CS exposure leads to the inflammation seen in COPD in the hope of discovering

novel, effective anti-inflammatory therapies to tackle underlying pathogenic disease

mechanisms.

Cytokines are thought to be critical orchestrators of the persistent inflammation seen in

several inflammatory diseases including COPD [3]. Many of these cytokines signal through

the Janus Kinase/ Signal Transducers and Activators of Transcription (JAK/STAT) pathway

and/or are produced as a result of JAK/STAT pathway signalling [4].

In mammals, there are 4 JAK family members: JAK1, JAK2, JAK3 and TYK2 and 7 STAT

family members: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. Binding

of a cytokine to its receptor activates the appropriate pair of JAK subunits which

phosphorylate the receptor cytoplasmic domain creating binding sites for STAT proteins.

Once bound the STAT proteins are phosphorylated at the tyrosine residue and then form

homo- or hetero-dimers, which rapidly migrate to the nucleus and act as transcription factors

to mediate gene expression [5–7].

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The JAK/STAT pathway has been targeted by several companies for treatments for a wide

range of inflammatory diseases. For example, inhibitors of the pathway have been approved

for the treatment of rheumatoid arthritis [8, 9]. In the respiratory field, the JAK/STAT

pathway has been implicated in the pathogenesis of asthma [10, 11]. There is currently

considerable interest in developing JAK/STAT inhibitors as a potential treatment for COPD.

However, there are no published studies which have comprehensively investigated the

activation of all the STAT family members in tissue from COPD patients.

Therefore the aim of this study was to determine the extent of JAK/STAT pathway activation

by detecting the presence of phosphorylated STAT proteins 1-6 in lung tissue from non-

smokers, smokers (with no apparent lung disease) and COPD patients by western blot and

immunohistochemistry (IHC). This will provide important target validation data regarding

the role of the JAK/STAT pathway in COPD which may be useful in the development of

more specific novel treatments for COPD.

3

Materials and Methods

The current study used human parenchymal samples from non-smokers, smokers (with no

apparent lung disease) and COPD patients assessing phosphorylation of STAT proteins as a

marker of JAK/STAT pathway activation by western blot and IHC.

Generation of human samples

Human donor and recipient lung tissue samples, surplus to clinical requirements, were

obtained from a transplant programme supported by the NIHR Respiratory Disease

Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and

Imperial College London. Informed written consent and ethical approval was obtained.

Samples were divided into 3 groups: donor (non-smokers), donor (smokers, with no apparent

lung disease) and COPD patients. Lung function data was available for 6 of the COPD

patients: average FEV1 of 0.54 (0.38-0.68) L, or 18.6 (15-26) % predicted, FVC 2.09 (1.11 –

2.62) L, all subjects were GOLD stage III. Limited information on smoking history was

available, the average pack history for the smoking donor group was 27.8 (14-37) years (n =

4) and COPD group was 47.6 (30-80) years (n = 5). None of the COPD patients were active

smokers at the time of the lung transplant.

Table 1. Subject demographics

Donor (non-smoker) Donor (smoker) COPD

n 12 10 23

Age 50.1 ± 13.5 39.8 ± 13.1 55.3 ± 4.6

4

Gender 3M:9F 7M:3F 7M:16F

Investigating STAT phosphorylation by western blot

Protein was extracted from frozen human lung parenchymal samples in the presence of

protease and phosphatase inhibitors and standard western blotting techniques were used for

the investigation of STAT phosphorylation. For an explanation of the full methods please see

the online supplement. Primary antibodies were purchased from New England Biolabs,

Hitchin, UK and used at a 1:500 dilution unless stated: phospho-STAT-1 (9167), phospho-

STAT-2 (4441), phospho-STAT -3 (9131), phospho-STAT -4 (5267), phospho-STAT -5

(9351), phospho-STAT -6 (9364), STAT-1 (9172), STAT-2 (4594), STAT-3 (9132), STAT-4

(2653), STAT-5 (9363), STAT-6 (9362), β-actin (A5060; Sigma-Aldrich Ltd., Poole, UK;

1:1000 dilution). Antibodies for phosphorylated ser 727 on STAT1 (9177S) and STAT3

(9134S) were purchased from New England Biolabs. A HRP-conjugated goat anti-rabbit

secondary antibody was used (P0448; Dako, Ely, UK; 1:5000 dilution). Band density of the

phospho-STAT proteins were analysed relative to density of the relevant STAT protein or the

loading control, actin, using ImageJ software.

Investigating STAT phosphorylation by IHC

Standard IHC techniques were used for the investigation of STAT phosphorylation. For an

explanation of the full methods please see the online supplement. Primary antibodies for IHC

were purchased from New England Biolabs unless stated: phospho-STAT-1 (9167), phospho-

STAT-2 (4441), phospho-STAT-3 (9131), phospho-STAT-4 (71-7900; Invitrogen) phospho-

5

STAT-5 (9359), phospho-STAT-6 (9364). A donkey anti-rabbit secondary antibody was

purchased from Jackson Immunoresearch, Newmarket, Suffolk, UK (1:200 dilution).

The intensity of the phospho-STAT staining in each sample was scored by eye using a light

microscope. Two independent observers, blinded to the sample group to avoid bias, scored

the slides. For each slide, the staining intensity was graded relatively based on the following

scale: 1, 2, 3 and 4 from no staining to strong brown staining. A score was recorded for the

staining confined to the macrophages, epithelium and for the overall slide. Ten different

fields on each slide were scored, and the average score was calculated for each slide. An

average score of the scores for each of the two observers was then calculated.

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Results

In order to determine whether the JAK-STAT pathway is activated in smokers and COPD

patients compared to non-smokers, the tyrosine phosphorylation of STATs 1-6, as markers of

JAK-STAT pathway activation, were assessed by western blot and IHC. We were also able to

investigate whether the phosphorylation was localised to epithelial cells or macrophages, by

assessing the intensity of immunohistochemical staining in these particular cell types.

Determining phosphorylation of STAT1 by western blot and IHC in human parenchymal

samples from non-smokers, smokers and COPD patients

Western blotting revealed that tyrosine phosphorylation of STAT1 appears to be increased in

smokers and COPD patients compared to non-smokers although this only reached

significance in COPD patients indicating that the JAK-STAT1 pathway is activated in COPD

patients (Figure 1). We could not detect any difference in levels of phosphorylation at ser 727

in the three groups. This suggests that the STAT 1 activation was via JAK (data not shown).

Immunohistochemical staining for tyrosine phospho-STAT1 displayed a similar trend

towards a small increase in smokers and COPD patients compared to non-smokers but this

did not reach significance (Figure 2). Staining was stronger in macrophages than epithelial

cells across all three groups.



We could not detect any phosphorylated tyrosine in STAT2 in any of the groups by western

blot. However a significant increase in phospho- tyrosine-STAT2 staining was seen in the

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macrophages of smokers compared to non-smokers by IHC (Figure 3). This increase did not

reach significance in epithelial cells or the overall section and no such increase was seen in

COPD patients.



Similar to the results for phospho-STAT1, there was a significant increase in the amount of

phospho-tyrosine-STAT3 detected by western blot in COPD patients compared to non-

smokers (Figure 4). A trend towards an increase in smokers compared to non-smokers did not

reach significance. We could not detect any difference in levels of phosphorylation at ser 727

in the three groups. This suggests that the STAT 3 activation was via JAK (data not shown).

Despite these findings by western blot no increase in tyrosine phospho-STAT3 staining was

seen in COPD patients compared to non-smokers by IHC (Figure 5). There appeared to be a

small increase in phospho-STAT3 in macrophages specifically from smokers compared to

non-smokers but this did not reach significance.



Phosphorylated tyrosine in STAT4 could not be detected in any of the samples by western

blot and no difference in phospho-STAT4 staining by IHC could be detected across the

groups in either cell type (epithelial cells or macrophages) evaluated or the overall section

(Figure 6).

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Phosphorylated tyrosine in STAT5 could not be measured in any of the samples by western

blot. Differences in phospho-tyrosine-STAT5 staining by IHC between the groups were also

not detected (Figure 7).



Although we could measure phosphorylated tyrosine in STAT6 by western blot, there was no

difference between the groups (Figure 8). Very little phospho-tyrosine-STAT6 was observed

by IHC and there were no significant differences between the groups (Figure 9).

9

Discussion

There are currently no effective treatments to stop the progression of COPD. Currently there

is interest in developing inhibitors of the JAK/STAT pathway as a novel treatment for COPD

as this pathway plays a key role in the signalling of several cytokines thought to drive the

persistent inflammation seen in COPD [3, 4]. Moreover, inhibitors of this pathway have been

approved for other inflammatory diseases such as rheumatoid arthritis [8, 9]. We wanted to

investigate which STAT proteins are activated in COPD by both western blot and IHC as

surprisingly there are no published studies showing the activation status of all STATs1-6 in

lung samples as compared to non-smoker and healthy smoker controls. While prospective

patient selection enables the collection of data from carefully phenotyped patients, BAL,

sputum samples, biopsies data can often be compromised by the small sample sizes, limited

area sampled and in some cases artefacts induced by the sampling techniques. For these

reasons it is important to carry out work in lung tissue in which larger areas can be sampled

and tissues can be preserved using methodologies which have less impact on assay protocols.

Importantly, the use of transplant tissue also allows us to use tissue from end stage COPD

patients who would not normally be consented for invasive procedures as part of a clinical

trial.

To our knowledge, this is the first study to show increased phosphorylation of tyrosine on

STAT1 in lung parenchymal samples taken from COPD patients compared to smokers

without COPD and non-smokers. This is particularly interesting as a recent genome wide

association study using a Norwegian cohort suggests that a polymorphism in the STAT1 gene

may confer susceptibility to developing COPD [12]. Equally, we were able to show increased

phosphorylation of STAT3 by western in COPD patients compared to non-smokers. This

finding is consistent with elevated levels of IL-6 that are seen in the induced sputum and lung

tissue of COPD patients [13, 14] and the fact that IL-6 is recognised to activate STAT3 [7].

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Interestingly, we could not detect any phosphorylation of Ser 727 on either STAT1 or

STAT3. As these sites are not thought to be phosphorylated by JAK it may imply that the

activation of STATs we observes is specific to JAK signalling. These data may suggest that

whilst the JAK/STAT pathway is involved in the pathogenesis of COPD only certain STATs

are involved. Therefore the possibility exists to target specific STAT proteins rather than

broad-spectrum inhibition which may increase the side effect liability.

Whilst the results obtained by IHC did not entirely match the results obtained by western

blot, the differences in results may simply be explained by the different protocols employed.

For example, fixing the lung tissue in formalin before IHC can mask epitopes of the target

proteins by crosslinking proteins or/and alter their tertiary structure [15], although steps were

taken to minimise this and unmask the epitopes. Alternatively the discrepancy in the results

may be due to differences in antibody affinity when utilised for each technique. Indeed,

phosphorylated STAT2 was found to be increased by IHC in macrophages from smokers

compared to non-smokers but we could not detect any phosphorylated tyrosine STAT2 in any

of the samples by western.

Apart from the increase in STAT2 activation in macrophages, no significant differences in

STAT activation between the groups were detected by IHC. Whilst we were able to show an

increase in STAT3 phosphorylation by western in COPD patients compared to non-smokers,

our IHC results did not illustrate this. Intriguingly, Ruwanpura and colleagues were only able

to demonstrate an association between activation of STAT3 and inflammation but not COPD

status when comparing COPD patients to ‘healthy’ smokers using IHC [14]. Whilst this

appears to support our data showing no difference in phosphorylated STAT3 staining

between COPD patients and smokers, it suggests that as COPD patients display airway

inflammation but non-smokers should not, we should have observed a difference between

11

these two groups. This suggests that the antibody in this case was more suitable for Western

blotting rather than IHC, or that the dynamic range for two different assays are different.

Increased phospho-STAT4 staining by IHC in smokers and COPD patients compared to non-

smokers has been reported previously by Di Stefano and colleagues [16]. Whilst we used the

same antibody we were unable to replicate their finding. This could be due to differences in

the general protocol or the samples employed. The previous paper used samples from patients

with mild or moderate COPD [16] whereas the samples in our study were from a transplant

programme and therefore the patients had end stage disease. It may be that STAT4 is only

activated during the early stages of COPD rather than the later stages.

There are limitations associated with the samples utilised for this study, which although

unavoidable here, should be addressed in future investigations. Firstly, equal numbers of

samples were not utilised in each group with a maximum of 12 non-smokers or 10 smokers

samples compared to 23 COPD patients samples. As mentioned previously the COPD

patients are at the end stage of the disease (the FEV1 data available from 6 of the COPD

patients indicated an FEV1 of 18.6 ± 1.7% predicted) and they have stopped smoking which

may affect the inflammatory status of their lungs. This is may explain why an increase in

STAT2 activation in macrophages was only observed in smokers but not COPD patients

compared to donor non-smokers. The COPD patients in our study would also have been

taking a range of differing medications depending on their exact disease phenotype. Although

the efficacy of these medications is controversial, they may affect the JAK STAT pathway

via effects on cytokine production. A larger study with equal groups would be needed to

validate these initial findings. It would also be interesting to assess STAT phosphorylation at

the different GOLD stages of COPD to investigate how the STAT proteins are involved in

disease progression and if different STATs are involved at different stages.

12

In summary, the results of this study suggest a role for the JAK/STAT pathway in COPD and

STAT1 and 3 in particular warrant further investigation as targets for novel COPD

therapeutics.

13

Figure Legends

Figure 1: STAT1 phosphorylation in human parenchymal samples from non-smokers,

smokers and COPD patients by western blot

Top panel: Representative image of western blot for phospho-STAT1, STAT1 and β-actin as

a loading control. D, donor; S, smoker; C, COPD.

Bottom panels: The level of phospho-STAT1 was measured by western blot relative to the

loading control, actin (left panel) and total level of STAT1 (right panel) in human

parenchymal samples from donor non-smokers, smokers and COPD patients. Data is

presented as mean ± SEM (AU) of densitometry readings. Statistical significance was

assessed using a Kruskal-Wallis test and Dunn’s multiple comparison post-test, *p<0.05 vs.

donor.

Results are representative of 12 non-smokers, 10 healthy smokers and 22 subjects with

COPD.

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smokers and COPD patients by IHC

A] Parenchymal sections from donor non-smokers, smokers and COPD patients were stained

for phospho-STAT1 by IHC. The level of staining in epithelial cells, macrophages and the

overall section was assessed on a scale from 1 to 4 by two independent observers. Ten fields

per section were assessed and an average was calculated. Data is presented as the mean ±

SEM. Epi, epithelial cells; Macs, macrophages.

Representative photomicrographs of a parenchymal section immunostained for identification

of phospho‐STAT1 from a donor (B), smoker (C) and COPD (D) patients. Scale bar = 100

µm. The arrow indicates positive staining.


COPD.

15







SEM. Statistical significance was assessed using a Kruskal-Wallis test and Dunn’s multiple

comparison post-test, *p<0.05 vs. donor. Epi, epithelial cells; Macs, macrophages.



µm.

16


COPD.








presented as mean ± SEM (AU) of densitometry readings. Statistical significance was


donor.

17


COPD.










µm.

18


COPD.










µm.

19


COPD.










µm.

20


COPD.








presented as mean ± SEM (AU) of densitometric readings. Statistical significance was


donor.

21


COPD.










µm.

22


COPD.

23

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