Therapeutic targeting of the IL-6 trans-signalling/mTORC1 axis in pulmonary

emphysema

Saleela M. Ruwanpura1,2, Louise McLeod1,2, Lovisa F. Dousha3, Huei J. Seow3, Sultan

Alhayyani1,2, Michelle D. Tate1,2, Virginie Deswaerte1,2, Gavin D. Brooks1,2, Steven

Bozinovski3,4, Martin McDonald5, Christoph Garbers6, Paul T. King1,5, Philip G. Bardin1,5,

Ross Vlahos3,4, Stefan Rose-John6, Gary P. Anderson3 and Brendan J. Jenkins1,2*

1Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research,

27-31 Wright Street, Clayton, Victoria, 3168, Australia.

2Department of Molecular Translational Science, Faculty of Medicine, Nursing and Health

Sciences, Monash University, Clayton, Victoria, 3800, Australia.

3Lung Health Research Centre, Department of Pharmacology and Therapeutics, The

University of Melbourne, Parkville, Victoria, 3050, Australia.

4School of Health and Biomedical Sciences, RMIT University, Bundoora, Victoria, 3083,

Australia.

5Monash Lung and Sleep, Monash Medical Centre, Victoria, 3168, Australia.

6Institute of Biochemistry, Christian-Albrechts-University, Olshausenstrasse 40, D-24098

Kiel, Germany.

*To whom correspondence should be addressed: Brendan J. Jenkins, Centre for Innate

Immunity and Infectious Diseases, Hudson Institute of Medical Research, 27-31 Wright

Street, Clayton, Victoria, 3168, Australia.

Tel +61 3 8572 2740; E-mail Brendan.Jenkins@hudson.org.au

Author contributions: S.M.R designed and performed experiments, analysed data, prepared

the figures and co-wrote the manuscript. L.M. performed mouse experiments, cell culture,

immunohistochemistry and molecular experiments. L.F.D. and H.J.S. performed smoke and

lung function studies. M.D.T. and V.D. performed cell culture and flow cytometry analyses,

respectively, and S.A. and G.D.B. assisted with immunohistochemistry. P.B. and P.T.K.

provided human lung biopsies, and M.M. provided human sera. G.P.A. assisted with the

experimental design of smoke-related studies and editing of the manuscript. R.V. and S.B.

analyzed data, and assisted with experimental design of smoke-related studies and editing of

the manuscript. S.R-J. provided mouse strains and novel reagents to target IL-6 trans-

signalling, and assisted with the experimental design and editing of the manuscript. B.J.J.

conceived, designed and supervised the research, and co-wrote the manuscript.

Sources of support: This work was supported by a research grant from the National Health

and Medical Research Council of Australia (NHMRC) awarded to B.J.J., as well as the

Operational Infrastructure Support Program by the Victorian Government of Australia. S.M.R.

is supported by a NHMRC Post-doctoral Training Fellowship. The work of S.R-J. was

supported by the Deutsche Forschungsgemeinschaft Bonn, Germany (SFB877, project A1 and

the Cluster of Excellence 'Inflammation at Interfaces'). B.J.J. was supported by a Senior

Medical Research Fellowship awarded by the Sylvia and Charles Viertel Charitable

Foundation, and is currently supported by an NHMRC Senior Research Fellowship. S.R-J. is

an inventor of patents owned by CONARIS Research Institute, which develops the sgp130Fc

protein together with Ferring Pharmaceuticals, and he has stock ownership in CONARIS. No

conflicts of interest, financial or otherwise, are declared by all other authors.

Running title: IL-6 trans-signalling/mTORC1 axis in emphysema

Descriptor number: 9.19

Word count: 3723

At a Glance Commentary:

Scientific Knowledge on the Subject

Emphysema is a major pathological component of COPD, and there is an urgent need for new

intervention strategies against this disease. The potent immunomodulatory cytokine IL-6 is a

prominent biomarker for emphysema. However, IL-6 is highly pleiotropic, and targeting IL-6

has been problematic since both pathogenic and beneficial (homeostatic) actions are blocked

concurrently by conventional anti-IL-6 antibodies. It has thus proven difficult to exploit IL-6

as a therapeutic modality in human emphysema.

What this Study Adds to the Field

Our work addresses a new area of IL-6 biology that exploits understanding differential modes

of IL-6 signalling, and their applicability to promoting the molecular pathogenesis of

emphysema. Specifically, IL-6 utilizes two distinct signalling modes via the gp130 signal-

transducing receptor subunit to regulate many pathophysiological responses, namely classical

signalling through the membrane-bound IL-6 receptor (IL-6R) subunit, and trans-signalling

via a naturally-occurring soluble IL-6R. In a novel combinatorial approach comparing the

lungs of emphysematous patients and mouse models for spontaneous (gp130F/F) and

experimentally-induced (cigarette smoke exposure) emphysema, we have elucidated the

specific contribution of IL-6 trans-signalling to emphysema. The knowledge generated by our

findings is crucial for the future design and clinical application of novel therapeutics against

emphysema.

"This article has an online data supplement, which is accessible from this issue's table of

content online at www.atsjournals.org"

1

ABSTRACT

Rationale: The potent immunomodulatory cytokine interleukin (IL)-6 is consistently

upregulated in human lungs with emphysema, and in mouse emphysema models; however,

the mechanism(s) by which IL-6 promotes emphysema remains obscure. IL-6 signals using

two distinct modes, classical signalling via its membrane-bound IL-6 receptor (mIL-6R), and

trans-signalling via a naturally-occurring soluble IL-6R (sIL-6R).

Objectives: To identify whether IL-6 trans-signalling and/or classical signalling contribute to

the pathogenesis of emphysema.

Methods: We utilized the gp130F/F genetic mouse model for spontaneous emphysema, and

cigarette smoke-induced emphysema models. Emphysema in mice was quantified by various

methods including in vivo lung function and stereology, and TUNEL assay was employed to

assess alveolar cell apoptosis. In mouse and human lung tissues, the expression level and

location of IL-6 signalling-related genes and proteins were measured, and the levels of IL-6

and related proteins in sera from emphysematous mice and patients were also assessed.

Measurements and Main Results: Lung tissues from emphysema patients, as well as from

spontaneous and cigarette smoke-induced emphysema mouse models, were characterized by

excessive production of sIL-6R. Genetic blockade of IL-6 trans-signalling in emphysema

mouse models, and therapy with the IL-6 trans-signalling antagonist sgp130Fc, ameliorated

emphysema by suppressing augmented alveolar type II cell apoptosis. Furthermore, IL-6

trans-signalling-driven emphysematous changes in the lung correlated with mammalian target

of rapamycin complex 1 (mTORC1) hyper-activation, and treatment of emphysema mouse

models with the mTORC1 inhibitor rapamycin attenuated emphysematous changes.

Conclusions: Collectively, our data reveal that specific targeting of IL-6 trans-signalling may

represent a novel treatment strategy for emphysema.

Abstract word count: 250

2

Key words: pulmonary emphysema, interleukin-6, mammalian target of rapamycin complex

1, mouse models, trans-signalling

3

INTRODUCTION

Pulmonary emphysema is the major component of Chronic Obstructive Pulmonary Disease

(COPD), a condition predicted to be the third-leading cause of death worldwide by 2020 (1).

The primary cause of emphysema is cigarette smoke (CS), which triggers a deregulated

immune response in the lung that drives the destructive loss of alveolar cells and airspace

enlargement (2). Despite these observations, the molecular basis by which key modulators of

the immune system promote emphysema remains unclear. In this regard, increased expression

of the immunomodulatory cytokine interleukin(IL)-6 is a common feature of emphysema, and

increased IL-6 expression and IL6 gene polymorphisms correlate with rapid lung function

decline and increased disease risk in smokers (3-6). Furthermore, elevated IL-6 expression in

various mouse emphysema models has been linked to disease pathogenesis (7-11).

IL-6 utilizes two distinct signalling modes, both dependent upon the essential and

ubiquitously-expressed gp130 cytokine receptor signal-transducing β-subunit, to regulate

many pathophysiological responses. IL-6 classical signalling occurs via its membrane-bound

(m) IL-6 receptor (mIL-6R) (12), and IL-6 trans-signalling (IL-6TS) via a naturally-occurring

soluble (s) IL-6R (13) produced either by alternative splicing of IL6R mRNA or proteolytic

cleavage of mIL-6R, the latter involving metalloproteases ADAM10 and/or ADAM17 (14).

Notably, IL-6TS enables a large array of mIL-6R- cell types to respond to IL-6 (12), thus

increasing the overall IL-6 signalling spectrum. The predominant signalling pathway of either

mode is JAK/STAT3, with other pathways including PI3K/AKT, mTORC1, and ERK/MAPK

(15, 16).

The broad array of cell types responsive to IL-6TS underpins its role as the likely

pathogenic IL-6 signalling mode in various inflammatory conditions such as arthritis, asthma,

Crohn’s disease, and sepsis (17-22), as well as numerous cancers (23-25). However, despite

these observations, the causal involvement of specific IL-6 signalling modes in emphysema is

4

unknown. We have previously reported the spontaneous development of IL-6-driven alveolar

cell apoptosis leading to emphysema in gp130F/F mice (11), which have been engineered to

display IL-6/gp130-dependent hyper-activation of the JAK-mediated STAT and mTORC1

pathways, in the absence of SHP2-mediated ERK MAPK and PI3K/AKT signalling. Here, we

reveal in both gp130F/F and CS-induced emphysema models that the specific blockade of IL-

6TS with the antagonist sgp130Fc (26) suppresses alveolar type II (ATII) cell apoptosis and

disease pathogenesis. Furthermore, we uncover a novel role for mTORC1 hyper-activation in

facilitating IL-6TS-driven emphysematous changes in the lung. Notably, the IL-

6TS/mTORC1 axis was also augmented in patients with emphysema. Collectively, our

observations provide the rationale for the clinical evaluation of IL-6TS as a new target for the

treatment of emphysema.

5

METHODS

Mice and treatments

The gp130F/F and gp130F/F:Il6-/- mice have been previously reported (11), and

gp130F/F:sgp130Fctg/tg mice were generated using sgp130Fc transgenic mice (27). Gp130F/F

mice aged 3 months were intraperitoneally (i.p) administered with either sgp130Fc (once

weekly, low dose of 0.5mg/kg) (21), PBS (once weekly), rapamycin (3 times a week, 4mg/kg)

(36) or vehicle (0.8% DMSO, 5.2% PEG-400, 5.2% Tween-20, 3 times a week), over 12

weeks. For acute and chronic CS models (details are provided in the online data supplement),

mice were concurrently administered with sgp130Fc (0.5mg/kg) or PBS once over 4 days and

once weekly over 12 weeks, respectively. Rapamycin (4mg/kg) or vehicle was administered

either twice over 4 days (acute CS) or 3 times a week over 12 weeks (chronic CS). All mice

were housed under specific pathogen-free conditions. Experiments were approved by the

Monash University Animal Ethics Monash Medical Centre “A” Committee“.

Human lung biopsy and serum samples

Details are provided in an online data supplement.

RNA isolation and gene expression analysis

Details are provided in an online data supplement.

Protein extraction, ELISA and immunoblotting

Details are provided in an online data supplement.

Immunohistochemistry and immunofluorescence

Details are provided in an online data supplement.

6

Stereology and mean linear intercept (MLI) analyses

Mouse lungs were perfusion fixed by instillation of 1.5% glutaraldehyde/1.5% formaldehyde

in 0.04M phosphate-buffered saline, following which tissue blocks were prepared by

processing and embedding in glycol methacrylate, as previously described (11). Lung

stereology was performed on methylene blue-stained lung tissue sections using computer-

assisted newCAST software (version 2.14; Visiopharm, Hørsholm, Denmark (7, 11). Airspace

enlargement was quantified by the MLI technique on H&E-stained lung sections (7).

Lung function analyses

The assessment of lung function on anesthetized mice was performed using the flexiVent

system (SCIREQ, Montreal, Canada) (7, 11).

Statistical analyses

Statistical analyses were performed using GraphPad Prism for Windows version 6.0.

D'Agostino and Pearson omnibus K2 normality tests were performed for all data. Paired t-

tests were used to analyze normally distributed data among 2 sample groups, and Mann-

Whitney tests for non-normally distributed data or smaller data sets. One-way analysis of

variance (ANOVA) was used to assess differences between 3 or more groups for normally

distributed data, and Kruskal-Wallis tests for non-normally distributed data or smaller data

sets, along with appropriate post-tests. Linear regression was performed for correlations

between given markers in moderate emphysema patient groups only. Data are expressed as

the mean ± standard error of the mean (SEM), and P < 0.05 was considered statistically

significant.

7

RESULTS

Augmented expression of IL-6TS components in gp130F/F emphysematous mouse lungs

To delineate the mode of IL-6 signalling associated with IL-6-driven emphysema, we initially

assessed whether key IL-6TS components were deregulated in emphysematous lungs of 6

month old (mo) gp130F/F mice. While gene expression levels for Il6rα (11) and Adam10 were

normal, Il6 (11) and Adam17 were significantly increased in gp130F/F mouse lungs (Fig. 1A).

IL-6 protein levels were also significantly increased in the lungs and bronchoalveolar lavage

fluid (BALF) of gp130F/F compared to gp130+/+ control wild-type mice (Fig. 1B, Fig. E1A).

Since sgp130 protein levels were unchanged, the high sIL-6R levels in lungs and BALF of

gp130F/F mice (Fig. 1B, Fig. E1A) suggest augmented IL-6TS and its possible role in the

emphysema phenotype of gp130F/F mice.

Transgenic over-expression of the IL-6TS antagonist sgp130Fc in gp130F/F mice

prevents the onset of emphysema

To define a causative role for IL-6TS in the pathogenesis of emphysema in gp130F/F mice, we

crossed gp130F/F mice with sgp130Fc transgenic mice (27) to generate gp130F/F:sgp130Fctg/tg

mice over-expressing sgp130Fc, a fusion protein of recombinant sgp130 and the Fc region of

human IgG1 which acts as a potent specific inhibitor of IL-6TS-driven pathologies (21, 24).

Histological evaluation of 6mo gp130F/F:sgp130Fctg/tg mice revealed normal alveolar lung

architecture compared to enlargement of the distal air spaces and destruction of alveoli,

measured as alveolar mean linear intercept (MLI), that were observed for gp130F/F mice (Fig.

1C, Fig. E1B). Consistent with these histological observations, the elevated static compliance

and lung volume, due to destruction of elastic fibres in emphysematous lungs, that are a

feature of gp130F/F mice (11) were also normal in gp130F/F:sgp130Fctg/tg mice (Fig. 1D, E). In

further support of these findings, stereology indicated that the volume fractions of air space

8

and alveolar septal tissue, and the surface density within lung parenchyma, were similar

between gp130+/+, gp130+/+:sgp130Fctg/tg and gp130F/F:sgp130Fctg/tg mice, while these

parameters were significantly elevated in gp130F/F mice (Table E1). The serum and lung

expression levels of the acute phase response (APR) protein serum amyloid A, which is

regulated by IL-6 classical signalling, remained elevated and unchanged in

gp130F/F:sgp130Fctg/tg compared to gp130F/F mice (Fig. E1C), thus indicating that IL-6

classical signalling was unaffected in gp130F/F:sgp130Fctg/tg mice. Furthermore, the absence

of emphysema in gp130F/F:sgp130Fctg/tg mice was associated with a significant reduction in

the lung expression levels of IL-6 and sIL-6R back to those observed in

gp130+/+:sgp130Fctg/tg and gp130+/+ mice (Fig. E1D, E). Collectively, these data reveal that

IL-6TS drives the development of emphysema in gp130F/F mice.

IL-6TS promotes apoptosis-driven emphysema in gp130F/F mice

Since apoptosis is a key process associated with the development of human emphysema and

several disease models, including IL-6/gp130-driven emphysema in gp130F/F mice (11, 28,

29), we compared the extent of apoptotic TUNEL-stained cells in the lungs of 6mo mice. The

number of TUNEL-stained alveolar cells in gp130F/F:sgp130Fctg/tg mice was significantly

reduced compared to gp130F/F mice, and was comparable to gp130+/+ and

gp130+/+:sgp130Fctg/tg control mice (Fig. 2A) (7, 11). The alveolar epithelium covers >99%

of the internal surface area of the lung and is mainly composed of ATI and ATII cells, the

latter of which uniquely synthesize and secrete surfactants, including the biomarker surfactant

protein C (SPC) which prevents small airway closure and alveolar collapse, thereby

maintaining normal lung structure (30, 31). Notably, the number of SPC-positive cells in the

lungs of gp130F/F mice was significantly reduced compared to gp130+/+ mice, whereas SPC-

positive cell numbers remained elevated in the lungs of gp130F/F:sgp130Fctg/tg mice (Fig. 2B).

9

Dual immunofluorescence staining with cleaved caspase-3 (marker of apoptosis) and SPC

further confirmed that there was a significant increase in the number of apoptotic SPC-

positive ATII cells in gp130F/F mice compared to gp130+/+, gp130+/+:sgp130Fctg/tg and

gp130F/F:sgp130Fctg/tg mice (Fig. 2C), while augmented apoptosis was not detected for ATI

(podoplanin-positive) and endothelial (CD31-positive) cells (Fig. E2). Collectively, these data

suggest that IL-6TS promotes type II cells to undergo apoptosis during emphysema.

Therapeutic blockade of IL-6TS with sgp130Fc prevents alveolar cell death and

emphysema in gp130F/F mice

To determine whether IL-6TS can serve as a bona fide therapeutic target for emphysema, we

next explored whether suppression of IL-6TS upon administration with sgp130Fc would

prevent emphysema development in gp130F/F mice from 3 months of age onwards. At the

completion of 12 weeks treatment with sgp130Fc, the lungs of gp130F/F mice were

comparable to those of untreated gp130+/+ mice, with fully-preserved lung morphology (Fig.

3A), static compliance (Fig. 3B), lung volume (Fig. 3C), stereological parameters (Table E1)

and the number of apoptotic cells (Fig. 3D). Taken together these data demonstrate that

therapeutic targeting of IL-6TS can prevent emphysema development in gp130F/F mice.

Acute CS exposure of wild-type mice augments expression of IL-6TS components which

is associated with elevated alveolar cell apoptosis

Since CS is the major trigger of emphysema in humans, we next assessed whether our

mechanistic findings for spontaneous IL-6TS-driven emphysema are relevant to CS-induced

lung disease. Upon an initial exposure of 4-6 week old gp130+/+ mice to an acute (4 day) CS

model, mRNA levels for Il6 (11) and Adam17 were significantly increased, while those for

Il6rα and Adam10 remained normal, compared to non-CS-exposed control mice (Fig. 4A).

10

Acute CS exposure also significantly augmented IL-6 and sIL-6R, but not sgp130, protein

levels (Fig. 4B). By contrast, the lungs of gp130+/+:sgp130Fctg/tg mice exposed to CS showed

no changes in Il6 and Adam17 mRNA levels (Fig. E3A), nor sIL-6R and sgp130 levels (IL-6

levels remained undetectable) (Fig. E3B). The response of gp130+/+ mouse lungs to CS

exposure was confirmed by CS-induced increases in whole lung mRNA levels of

inflammatory mediators Cxcl2, Tnfα and Ccl2 (32, 33), which were ameliorated in either CS-

exposed gp130+/+:sgp130Fctg/tg mice or CS-exposed gp130+/+ mice concurrently administered

once with sgp130Fc (Fig. E3C). Moreover, while acute CS exposure of gp130+/+ mice also

augmented numbers of TUNEL-positive apoptotic alveolar cells (Fig. 4C) (11), no such

increases were observed in acute CS-exposed gp130+/+:sgp130Fctg/tg mice and CS-exposed

gp130+/+ mice treated with sgp130Fc (Fig. 4C).

Inhibition of IL-6TS with sgp130Fc suppresses alveolar cell apoptosis and

emphysematous changes induced by chronic CS exposure of wild-type mice

To determine the relevance of IL-6TS to CS-induced emphysema development, we next

utilized a chronic (12 week) CS-induced emphysema model. Over 12 weeks, we observed

significant increases in numerous parameters indicative of emphysema, namely static

compliance, lung volume and MLI, in CS-exposed gp130+/+ mice compared to their age-

matched non-CS-exposed counterparts (Fig. 4D-F). By contrast, chronic CS-exposed mice

displayed significant reductions in all of these emphysematous parameters upon inhibition of

IL-6TS with sgp130Fc (Fig. 4D-F). Importantly, these findings correlated with our data

demonstrating that chronic CS exposure increased TUNEL-positive cell numbers in the lungs

of gp130+/+ mice compared to non-CS-exposed mice, and this was significantly reduced

(~40%) in response to sgp130Fc treatment (Fig. 4G).

11

Collectively, these data in CS-exposed wild-type mice reveal that therapeutic and

genetic blockade of IL-6TS suppresses augmented expression of inflammatory mediators and

alveolar cell death, leading to amelioration of emphysema, therefore validating the relevance

of IL-6TS to the pathogenesis of CS-induced emphysema.

IL-6TS in the lungs of emphysematous gp130F/F mice is associated with hyper-activation

of the mTORC1 pathway

Our data here indicating that suppression of IL-6TS in mouse emphysema models prevents

emphysema implies that IL-6TS-driven emphysematous changes in the lung are a

consequence of increased activation of a gp130-dependent signalling pathway. We have

previously eliminated a role for IL-6-driven hyper-activation of STAT3 and STAT1 in

promoting emphysema in gp130F/F mice (7). In addition, such a pathway is unlikely to be

either SHP2-mediated ERK MAPK or PI3K/AKT, since gp130/SHP2-mediated activation of

these pathways has been abolished in gp130F/F mice (34). Another candidate pathway is

rapamycin-sensitive mTORC1, which is a large multi-protein complex comprising the

serine/threonine kinase mTOR subunit that is activated in response to diverse stimuli, such as

cytokines (35). Hyper-activation of mTORC1 is observed in many diseases, including

pulmonary fibrosis, which highlights its ability to regulate various cellular processes such as

proliferation and apoptosis (35, 36).

We observed a significant increase in the number of cells expressing phosphorylated

ribosomal S6 kinase (P-rpS6), a key mTORC1 substrate (35), in the lungs of gp130F/F mice

compared to emphysema-free gp130+/+ and gp130F/F:Il6-/- mice, the latter deficient in IL-6 (11)

(Fig. 5A). Dual immunofluorescence staining confirmed that P-rpS6 expression co-localized

to SPC-positive ATII cells (Fig. E4A), but not CD45-positive inflammatory cells (Fig. E4B).

Immunoblot analyses also indicated that P-rpS6 protein levels, as well as P-mTOR (ser2448)

12

levels, were significantly reduced in gp130F/F:Il6-/- compared to gp130F/F lungs (Fig. 5B, Fig.

E4C). Furthermore, we demonstrated activation of the IL-6TS/mTORC1 axis in cultured

gp130F/F primary ATII cells stimulated with the potent IL-6TS agonist Hyper-IL-6 (12) (Fig.

E5). Moreover, both the sgp130Fc-mediated genetic and therapeutic blockade of IL-6TS in

gp130F/F mice significantly reduced P-rpS6-positive cell numbers in the lung compared to

their respective controls (Fig. 5C, D, Fig. E6A, B). Therefore, these data support the notion

that IL-6TS augments activation of the mTORC1 pathway in the lungs of emphysematous

gp130F/F mice.

The therapeutic blockade of the mTORC1 pathway in rapamycin-treated gp130F/F mice

suppresses the development of emphysema

To specifically delineate a role for increased mTORC1 activity in IL-6TS-driven emphysema,

we explored whether mTORC1 pathway blockade with the specific mTORC1 inhibitor,

rapamycin, could alleviate emphysema development in gp130F/F and CS-induced emphysema

models. Examination of lung morphology, static compliance, lung volumes and various

stereological parameters revealed that emphysematous changes (Fig. 5E-G, Table E1), and

also TUNEL- and P-rpS6-positive cell numbers (Fig. 5H, I, Fig. E6C, D), were significantly

reduced in the lungs of gp130F/F mice following 12 weeks of rapamycin treatment compared

to vehicle-treated gp130F/F control mice.

We also observed increased numbers of P-rpS6-positive cells in the lungs of acute CS-

exposed gp130+/+ mice compared to their non-CS-exposed controls (Fig. E7A). However, P-

rpS6-positive cell numbers were significantly reduced in CS-exposed gp130+/+ mice

concurrently treated with sgp130Fc compared to CS-exposed gp130+/+ mice treated with

vehicle (Fig. E7A). Furthermore, the genetic blockade of IL-6TS in gp130+/+:sgp130Fctg/tg

mice prevented any increase in P-rpS6-positive cell numbers in response to CS exposure (Fig.

13

E7A). The numbers of P-rpS6- and TUNEL-positive cells were significantly reduced in acute

CS-exposed gp130+/+ mice administered with rapamycin compared to their CS-exposed

controls that received vehicle (Fig. E7B, C). Moreover, chronic CS exposure increased P-

rpS6-positive cell numbers in the lungs of gp130+/+ mice compared to their non-CS-exposed

counterparts, and this was significantly reduced by ~45% in response to sgp130Fc treatment

(Fig. 5J, Fig. E7D). Collectively, these data strongly suggest that IL-6TS promotes mTORC1

pathway hyper-activation during emphysema.

Upregulation of IL-6TS/mTORC1 components correlates with human emphysema

To translate our emphysematous mouse model findings to human disease, we first assessed

the expression of IL-6TS-related genes in human lung tissues from disease-free and

emphysema patients as defined by pulmonary lung function tests (gas exchange, FEV1; Table

E2). The gene expression level for IL6, as well as both IL-6 and sIL-6R protein levels, were

significantly increased in lungs of emphysema patients compared to emphysema-free controls

(Fig. 6A, B). In addition, ADAM17 gene expression was elevated 2-fold in emphysema

patients compared to disease-free individuals (Fig. 6A), and ADAM17 protein levels

significantly correlated with elevated sIL-6R levels observed in emphysema patients (Fig. 6C,

D). Furthermore, we observed a significant positive correlation between sIL-6R levels and the

number of cleaved caspase-3-positive cells in the lungs of emphysema patients (Fig. 6E, F),

thus further supporting IL-6TS-induced apoptosis in human emphysema.

We next investigated the activation status of the mTORC1 pathway, and immunoblot

analyses indicated that P-rpS6 protein levels were significantly increased in lung lysates from

emphysema patients (Fig. 6G). Furthermore, regression analyses of IL-6 and P-rpS6 levels

showed significant positive correlations with sIL-6R in emphysema patients (Fig. 6H).

Notably, circulating levels of IL-6TS components, namely IL-6 and sIL-6R, were also up-

14

regulated in the serum of emphysema patients compared to healthy individuals (Fig. 6I).

Overall these clinical data are consistent with our in vivo findings and provide translational

support for a pathological role for the IL-6TS/mTORC1 axis in human emphysema.

15

DISCUSSION

Despite the established link between IL-6 and the pathogenesis of emphysema and other lung

diseases (1-5, 11), little information is known regarding the identity of the mode of IL-6

signalling and downstream pathways which drive disease pathogenesis. For instance, mouse

asthma models and clinical data indicate increased sIL-6R levels in BALF and airway smooth

muscle which may be of relevance to airway remodelling (22, 37). In addition, blocking

global IL-6 signalling via neutralizing antibodies or genetic ablation of IL-6 during chronic

stages of adenosine-mediated lung injury and asthma provide the benefit of halting airway

remodelling processes such as fibrosis. However, the above studies only hypothesize

involvement of IL-6TS rather than IL-6 classical signalling in driving lung disease (38, 39).

Here, we define for the first time that hyper-activation of the endogenous IL-6TS/mTORC1

axis in the lung augments ATII cell apoptosis during emphysema. Furthermore, our

discoveries of augmented expression and activation of IL-6TS and mTORC1 pathway

components, respectively, in the lung of patients with emphysema, together with elevated

levels of IL-6TS components (e.g. sIL-6R) in the serum of emphysema patients, have

considerable translational potential for biomarker discovery and early disease detection, as

well as patient stratification for potential responders who may gain benefit from selective

therapies against the IL-6TS/mTORC1 axis.

While our data reveal that mTORC1 hyper-activation is associated with elevated

alveolar cell apoptosis, mTORC1 is also a negative regulator of autophagy, a process which

has been implicated in emphysematous lung destruction. However, we have previously

reported that IL-6-driven emphysematous changes in the lung (which are associated with

elevated mTORC1 activation) are independent of autophagy, and for that matter inflammation

(7). Furthermore, considering that mTORC1 is activated by a vast range of stimuli (e.g.

cytokines, growth factors, stress, oxygen, nutrients) and regulates a plethora of cellular

16

responses in the lung in both a cell-type and stimulus-dependent manner (35, 40), our current

findings suggest that mTORC1 activation in the alveolar epithelium specifically by IL-6

(unlike other stimuli) does not affect autophagy and/or inflammation.

Hyper-activation of mTORC1 can promote IL-6TS by upregulating sIL-6R levels in

vivo (41), which not only supports mTORC1 being an integral component of the IL-6TS

network (at least in the lung), but also suggests a feed-forward regulatory loop involving IL-

6TS and mTORC1. A role for mTORC1 in promoting emphysema is also supported by the

demonstration that peripheral blood mononuclear cells from COPD patients and CS extract-

stimulated human monocytes display increased mTOR activity, which is associated with

corticosteroid resistance in COPD patients (42). However, in contrast to our current findings,

expression of the CS-induced stress response protein, RTP801, can increase septal cell death

leading to pulmonary injury and emphysema via inhibition of mTORC1 and NF-κB activation

(43). Since the upstream activators of mTORC1 in the above study were not documented,

they may differ from IL-6 which could cause differences in the kinetics of mTORC1

activation and subsequently the net signal output and cellular response within the lung. In this

regard, mTORC1 can exhibit pro- and anti-apoptotic activities (35), and although the ability

of hyper-activated mTORC1 to promote apoptosis has been linked to a negative feedback

loop which suppresses the AKT survival pathway (35, 44), AKT activation in the lungs of

gp130F/F mice is unaltered (data not shown). Collectively, these observations not only

highlight the importance of identifying the molecular drivers and cellular context of mTORC1

activity during disease pathogenesis prior to the potential application of therapeutics, but also

warrant investigations into the existence of alternate signalling pathways (e.g. ERK MAPK)

and target genes (e.g. p53, Bcl-2, Nox4, Bad) which can promote the pro-apoptotic actions of

mTORC1 (45) during emphysema.

17

Current strategies aimed at targeting IL-6 in the clinic have largely resulted from earlier

experimentally-induced mouse models for arthritis and inflammatory bowel diseases,

whereby antibody–mediated neutralization of IL-6R or IL-6 suppressed disease development

(46). In this respect, the humanized neutralizing IL-6R monoclonal antibody, tocilizumab, has

shown considerable promise in clinical trials in the management of rheumatoid arthritis (47).

However, tocilizumab inhibits the mIL-6R mediated pathway, which most likely accounts for

the increased incidence of infection associated with its use due to suppressing the “immune-

protective” IL-6 classical signalling pathway. Indeed, studies using IL-6 knock-out mice have

suggested that IL-6 (classical) signalling protects against mucosal ulcerations from bacterial

pathogens, such as Citrobacter rodentium infection (48). Such studies highlight how, under

certain situations, blocking IL-6 (classical) signalling may have detrimental consequences,

and also imply that other modes of IL-6 signalling promote chronic inflammatory responses

associated with disease pathogenesis.

The specific IL-6TS antagonist sgp130Fc used in our current study can exhibit

significant therapeutic activity in IL-6TS-driven animal models of inflammation,

autoimmunity and cancer, with very low immunogenicity and ~7 day in vivo half-life, as well

as no interference with IL-6 classical signalling events (18, 21, 49). Notably, blocking IL-6TS

with sgp130Fc can inhibit sIL-6R production by macrophages contributing to attenuation of

idiopathic pulmonary fibrosis (50), which is consistent with our findings that sgp130Fc

suppressed augmented levels of sIL-6R in the lungs of gp130F/F and CS-induced emphysema

models. Moreover, the efficacy of sgp130Fc, which will undergo future phase II clinical trials

in Europe for Crohn’s Disease, at suppressing emphysematous changes in the lungs of both

models provides translational implications directly to preventatives for human emphysema.

From a therapeutic perspective the selective blockade of IL-6TS, and lung-directed mTORC1

suppression, offer promising new strategies to ameliorate emphysema (and COPD), and our

18

current findings will fast-track further pre-clinical testing of sgp130Fc and the development of

humanized anti-IL-6R mAbs which target IL-6TS for clinical use in emphysema.

19

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27

FIGURE LEGENDS

Figure 1 Augmented expression of IL-6TS components promotes emphysema in

gp130F/F mice. Q-PCR analyses of (A) Adam17 and Adam10 gene expression were performed

on cDNA derived from total RNA prepared from lung tissue of 6 month old (mo) gp130+/+

(+/+), gp130+/+:sgp130Fctg/tg (+/+:tg), gp130F/F (F/F) and gp130F/F:sgp130Fctg/tg (F/F:tg) mice.

Expression data are shown following normalization for 18S expression, and are presented as

the mean ± SEM from triplicate analyses from n = 5 mice per genotype. (B) IL-6, sIL-6R and

sgp130 protein concentrations in lung lysates as determined by ELISA. n = 5 mice per

genotype. (C) Representative methylene blue-stained cross-sections of lungs from 6mo +/+,

+/+:tg, F/F and F/F:tg mice. Arrows indicate airspace enlargement. Scale bars = 100μm. (D)

Static compliance as determined by flexiVent lung function analysis, and (E) lung volume per

body total weight (grams), of the above 6mo mice. Data are expressed as mean ± SEM. n =

10 mice per genotype. *P< 0.05, **P< 0.01, ***P< 0.001 and ****P< 0.0001.

Figure 2 IL-6TS-mediated apoptosis of ATII cells leads to the development of

emphysema. Stereological quantification of the number of (A) TUNEL- and (B) SPC-

positive ATII cells, and corresponding representative photomicrographs showing positive

cells in cross-sections of lungs in 6 month old (mo) gp130+/+ (+/+), gp130+/+:sgp130Fctg/tg

(+/+:tg), gp130F/F (F/F) and gp130F/F:sgp130Fctg/tg (F/F:tg) mice. Scale bars = 25μm. (C)

Representative confocal immunofluorescence photomicrographs of cells positive for either

caspase-3 alone (green), SPC alone (red), or both caspase-3 and SPC (yellow, white circles)

in cross-sections of the lung in 6mo F/F mice. The graph depicts stereological quantification

of dual labelled caspase-3- and SPC-positive cells in the lungs of 6mo mice of the indicated

genotypes. Scale bars = 10μm. Data are expressed as the mean ± SEM. n = 5 mice per

genotype. *P< 0.05 and **P< 0.01.

28

Figure 3 Therapeutic blockade of IL-6TS prevents emphysema development in

gp130F/F mice. (A) Representative methylene blue-stained cross-sections (arrows indicate

airspace enlargement), (B) static compliance and (C) lung volume, of lungs from 6 month old

(mo) gp130+/+ (+/+, n = 8) and gp130F/F (F/F, n = 8) mice, and 3mo F/F mice treated with

sgp130Fc inhibitor for 12 weeks (F/F+sgp130Fc, n = 5). Scale bars = 100μm. (D)

Stereological quantification of the number of TUNEL-positive cells, and corresponding

representative photomicrographs showing positive cells in cross-sections of lungs, from +/+

(n = 8), F/F (n = 8) and F/F+sgp130Fc (n = 5) mice. Data are expressed as the mean ± SEM.

*P< 0.05, **P< 0.01 and ***P< 0.001.

Figure 4 Blockade of IL-6TS with sgp130Fc suppresses apoptosis induced by CS

exposure to wild-type mice. (A) Q-PCR expression analyses of the indicated genes were

performed on lung cDNA, and (B) protein expression analyses of the indicated proteins were

performed on lung lysates, from 1 month old (mo) gp130+/+ (+/+) mice with (+) or without (-)

CS exposure for 4 days. Data are expressed as the mean ± SEM. n = 5 mice per genotype.

*P< 0.05. (C) Stereological quantification of the number of TUNEL-positive cells, and

corresponding representative photomicrographs showing positive cells in cross-sections of

lungs, from the following mouse groups; +/+ (- CS), +/+ (+ CS), +/+ concurrently treated

with sgp130Fc (+/+ (Fc + CS)) and gp130+/+:sgp130Fctg/tg (+/+:tg) mice with or without CS

exposure for 4 days. (D) Static compliance, (E) lung volume, (F) mean linear intercept, and

(G) stereological quantification of the number of TUNEL-positive cells and corresponding

representative photomicrographs showing positive cells in cross-sections of the lungs from

1mo gp130+/+ mice (+/+) treated with or without sgp130Fc inhibitor or CS for 12 weeks. Data

are expressed as the mean ± SEM. n = 5 mice per genotype. *P< 0.05, **P< 0.01, ***P<

0.001 and ****P< 0.0001.

29

Figure 5 IL-6-induced mTORC1 pathway hyper-activation promotes emphysematous

changes in the lungs of mice. (A) Stereological quantification of the number of P-rpS6-

stained cells, and corresponding representative photomicrographs showing positive cells in

cross-sections of lungs, from 6 month old (mo) gp130+/+ (+/+), gp130F/F (F/F) and F/F:Il6-/-

mice. n = 5 mice per genotype. *P< 0.05. (B) Immunoblotting with indicated antibodies on

whole lung lysates from 6mo F/F and F/F:Il6-/- mice. Each lane represents tissue from an

individual mouse. Densitometric quantification of P-rpS6 and total S6 protein levels per

genotype was performed and normalized against Actin protein levels present in each sample.

Data are presented as the mean fold induction ± SEM from n = 7 mice per genotype.

Stereological quantification of the number of P-rpS6-positive cells in the lungs of (C) 6

month old (mo) +/+, gp130+/+:sgp130Fctg/tg (+/+:tg), F/F and gp130F/F:sgp130Fctg/tg (F/F:tg)

mice and (D) 3mo F/F mice treated with sgp130Fc inhibitor for 12 weeks (F/F + Fc). n = 5

mice per group. *P< 0.05, **P< 0.01. (E) Representative methylene blue-stained cross-

sections of lungs from F/F mice (aged 1 month) that were treated with or without rapamycin

for 12 weeks. Arrows indicate airspace enlargement. Scale bars = 100μm. (F-I) Static

compliance (F), lung volumes (G), and the stereological quantification of the number of

TUNEL-positive cells (H) and P-rpS6-positive cells (I) in the lungs of F/F mice treated with

or without rapamycin for 12 weeks. n = 4 mice per group. *P< 0.05 and **P< 0.01. (J)

Stereological quantification of the number of P-rpS6-positive cells in the lungs of gp130+/+

mice (+/+) aged 1 month treated with or without sgp130Fc inhibitor or CS for 12 weeks. n =

4 mice per group. **P< 0.01 and ***P< 0.001.

Figure 6 Augmented levels and correlations of IL-6TS components in human

emphysema tissue and serum. (A) Q-PCR gene expression analyses of IL6 and ADAM17

were performed on human lung cDNA from emphysema patients (Emph, n = 20) or

30

emphysema-free individuals (Ctl, n = 10). Expression data are shown following

normalization for 18S expression, and are presented as the mean ± SEM from triplicate

analyses. (B) IL-6 and sIL-6R protein concentrations in lung lysates as determined by ELISA.

Data are expressed as the mean ± SEM. (C) Immunoblotting with antibodies against

ADAM17 and Actin on whole lung lysates from Emph and Ctl groups. Each lane represents

tissue from an individual. (D) Linear correlation between sIL-6R and ADAM17 (normalized

to Actin following densitometry) protein levels from individuals with emphysema (n = 9). (E)

Representative cross-section of the lung from an emphysema patient displaying cleaved

caspase-3-positive cells. Arrows indicate positive cells. Scale bar = 100μm. (F) The number

of cleaved caspase-3-positive cells per 20 high power fields in the lungs of individuals with

emphysema (n = 9) were measured, and then linear correlation was performed between sIL-

6R levels and the number of cleaved caspase-3-positive cells. (G) Immunoblotting with the

indicated antibodies on lung lysates from Emph and Ctl groups, and each lane represents

tissue from an individual. Densitometric quantification of P-rpS6 protein levels per group was

performed and normalized against Actin protein levels present in each sample. Data are

presented as the mean fold induction ± SEM. (H) Linear correlation between IL-6 and P-rpS6

protein levels compared to sIL-6R protein levels in lung tissue lysates from individuals with

emphysema (n = 20). (I) Protein levels of IL-6 and sIL-6R in the serum of Emph and Ctl

groups, as determined by ELISA. *P< 0.05 and **P< 0.05 versus Ctl group.

1

Therapeutic targeting of the IL-6 trans-signalling/mTORC1 axis in pulmonary

emphysema

Saleela M. Ruwanpura, Louise McLeod, Lovisa F. Dousha, Huei J. Seow, Sultan Alhayyani,

Michelle D. Tate, Virginie Deswaerte, Gavin D. Brooks, Steven Bozinovski, Martin

McDonald, Christoph Garbers, Paul T. King, Philip G. Bardin, Ross Vlahos, Stefan Rose-John,

Gary P. Anderson and Brendan J. Jenkins

"Online Data Supplement"

2

METHODS

Human lung biopsy and serum samples

Lung tissue from resection surgery for treatment of a solitary peripheral carcinoma was

collected from patients with evidence of emphysema, as defined by pulmonary function tests

(gas exchange, FEV1) (Table E2). Tissue from the subpleural parenchyma avoiding tumor-

bearing areas was either snap frozen for molecular analyses or formalin fixed by immersion

and paraffin embedded for histological analyses.

For serum collection, emphysema patients (n = 17) with moderate to severe stable disease

were defined by a TLCO (% of predicted post-oxygen transfer into the blood which determines

emphysema severity) of 36.2 ± 10.4 and FEV1 (% of predicted post-bronchodilator as a

measure of airflow obstruction which determines COPD severity) of 38.2 ± 12.7. These

patients also had radiological assessments such as high resolution computerized tomography

(CT) of the chest to verify emphysema. Control serum samples (n = 6) were donated by healthy

individuals with no emphysema. Lung tissue and serum were collected from individuals upon

written informed consent, and studies were approved by the Monash Health Human Research

Ethics Committee.

RNA isolation and gene expression analysis

Total RNA extraction and the preparation of cDNA for quantitative RT-PCR (Q-PCR)

expression analyses of individual genes were performed as previously described (1, 2).

Sequence information for primers against mouse genes 18s, Il-6, Il-6ra, Cxcl2, Ccl2 and Tnfa,

as well as the human gene 18S, have been previously published (1). Primer sequences for the

mouse gene Adam17 are; Adam17 forward 5’-GGCAACTCCAGGGTGGACGAAGGA-3’,

Adam17 reverse 5’-ATCTTCAGCATCTCCTGGTGGGGG-3’. Sequences for human primers

are as follows; IL-6R forward 5’-AAAGCTGGGCAGGTTGGTG-3’, IL-6R reverse 5’-

3

AGCTTGTGCAGAGGTGTTGAG-3’; ADAM17 forward 5’-

GAAGTGCCAGGAGGCGATTA-3’, ADAM17 reverse 5’-

GGGCACTCACTGCTATTACC-3’. Adam10/ADAM10 forward 5’-

CCTGAGCTCCTGAGGAAAA-3’ and Adam10/ADAM10 reverse 5’-

TGAGCAATCACAGCTTCTCG-3’ primer sequences were used for both mouse and human

genes.

Protein extraction, ELISA and immunoblotting

Total protein lysates were prepared from snap-frozen lung tissues (1, 2) and BALF, and were

subjected to ELISA. The mouse IL-6 and human IL-6 ELISA sets were purchased from BD

Sciences, (Franklin Lakes, NJ), while sIL-6R and sgp130 ELISA sets were purchased from R

& D systems (Minneapolis, MN). The SAA ELISA set was purchased from Life technologies

(Grand Island, NY). All ELISA sets were used as per the manufacturer’s instructions.

Immunoblotting of P-rpS6 (Ser240/244, Cell Signaling Technology (CST), Denver, MA), rpS6

(CST), P-mTOR (Ser2448, CST), mTOR (CST) and ADAM17 (Millipore, Temecula, CA)

were performed on lung lysates, and protein bands were visualized using the Odyssey Infrared

Imaging System (LI-COR, Lincoln, NE) and quantified using the Image J program (nih.gov).

Antibody against actin was purchased from Sigma-Aldrich (St. Louis, MO).

Immunohistochemistry and immunofluorescence

Apoptosis was determined by the terminal deoxynucleotidyl transferase (tdT)-mediated dUDP

nick-end labeling (TUNEL) technique using an ApopTag Peroxidase In Situ Apoptosis

Detection kit (Millipore, Billerica, MA). Immunohistochemistry with antibodies against SPC

(Santa Cruz Biotechnology, Dallas, Texas), P-rpS6 (Ser240/244, CST) and cleaved caspase-3

4

(CST) were performed as previously described (1). Stereological techniques were applied to

determine the number positively-stained alveolar cells.

Dual immunofluorescence staining was performed on paraffin-embedded lung tissues

using primary antibodies conjugated with fluorescent dyes or Alexa Fluor® secondary

antibodies. Briefly, lung sections were antigen retrieved by microwave in 1mM

Ethylenediaminetetraacetic acid (EDTA, pH8.0) buffer, following which they were blocked

with universal CAS block (Invitrogen, Carlsbad, CA) for 1 hour. Antigens were detected with

cleaved caspase-3 antibody conjugated with Alexa Fluor® 488 (CST), podoplanin antibody

conjugated with Alexa Fluor® 594 (BioLegend, San Diego, CA), CD31 antibody conjugated

with Alexa Fluor® 647 (BioLegend), P-rpS6 antibody conjugated with Alexa Fluor® 647

(CST), and SPC (Santa Cruz Biotechnology) and CD45 (BD BioSciences, Franklin Lakes, NJ)

primary antibodies with Alexa 488/546-conjugated donkey anti-goat and Alexa 488-

conjugated goat anti-rabbit secondary antibodies, respectively (Invitrogen). Slides were

examined under a Nikon confocal microscope and analyzed for red and green fluorescence.

Stereological techniques were applied to determine the number of dual stained cells.

Cigarette smoke (CS) exposure

Mice aged 4-6 weeks were subjected to whole body CS exposure for 4 days (acute) (1). This

smoke exposure protocol caused acute inflammation with no acute lung injury. For chronic CS

exposure, mice were subjected to whole body CS exposure over a 12 week period as per the

protocol described for acute CS exposure.

Primary lung cell cultures, and flow cytometry

Mouse primary alveolar type II (ATII) epithelial cells were prepared as previously described

(3). Following the digestion of lungs, single cell suspensions were prepared and then incubated

5

with purified CD45 antibody (BD Biosciences) and epithelial cells negatively enriched with

BioMag goat anti-rat immunoglobulin-coupled magnetic beads (Qiagen, Hilden, Germany).

The negative selection for ATI epithelial cells was performed by incubation of podoplanin-

coated petri dishes for 30 minutes at 370C. Cell purity was routinely assessed by epithelial cell

morphology and flow cytometry analysis with anti-EpCAM, anti-podoplanin and anti-SPC

(ATII) antibodies. Cells were cultured on collagen-coated (MP Biomedicals, Santa Ana, CA)

plates and stimulated with 100ng/ml Hyper-IL-6, an IL-6/soluble IL-6R fusion protein (4) over

the indicated times. Monolayers were detached with either 3mM EDTA for flow cytometry

analysis with Annexin-V (BD Biosciences) and 7-AAD to examine the level of apoptosis, or

RIPA buffer for immunoblot analyses of mTORC1 activity. Cells were analysed on a FACS-

CantoII flow cytometer (BD Biosciences) and data was performed using FACSDiva software

(BD Biosciences).

6

REFERENCES

1. Ruwanpura SM, McLeod L, Miller A, Jones J, Bozinovski S, Vlahos R, Ernst M, Armes J,

Bardin PG, Anderson GP, Jenkins BJ. Interleukin-6 promotes pulmonary emphysema

associated with apoptosis in mice. Am J Respir Cell Mol Biol 2011; 45: 720-730.

2. Ruwanpura SM, McLeod L, Miller A, Jones J, Vlahos R, Ramm G, Longano A, Bardin PG,

Bozinovski S, Anderson GP, Jenkins BJ. Deregulated Stat3 signaling dissociates

pulmonary inflammation from emphysema in gp130 mutant mice. Am J Physiol Lung

Cell Mol Physiol 2012; 302: L627-639.

3. Londrigan SL, Tate MD, Job ER, Moffat JM, Wakim LM, Gonelli CA, Purcell DF, Brooks

AG, Villadangos JA, Reading PC, Mintern JD. Endogenous murine BST-2/Tetherin is

not a major restriction factor of Influenza A virus infection. PLOS One 2015; 10:

e0142925.

4. Peters M, Muller AM, Rose-John S. Interleukin-6 and soluble interleukin-6 receptor: direct

stimulation of gp130 and hematopoiesis. Blood 1998; 92: 3495-3504.

7

SUPPLEMENTARY FIGURE LEGENDS

Figure E1: Expression of IL-6 signalling components in emphysematous gp130F/F and

emphysema-free gp130F/F:sgp130Fctg/tg mice. (A) IL-6, sIL-6R and sgp130 protein

concentrations were determined by ELISA in the BALF of 6 month old (mo) gp130+/+ (+/+)

and gp130F/F (F/F) mice. (B) Mean linear intercept of +/+, gp130+/+:sgp130Fctg/tg (+/+:tg), F/F

and gp130F/F:sgp130Fctg/tg (F/F:tg) mice aged 6 months. (C) Serum amyloid A (SAA) levels

in the serum and lungs of the indicated 6mo mice. Data are expressed as mean ± SEM. n = 5

mice per genotype. *P< 0.05, **P< 0.01 and ***P< 0.001. ND = not within assay detectable

level (assumed 0 for statistical analyses).

Figure E2: IL-6TS-mediated apoptosis is not observed in alveolar type I (ATI) and

endothelial cells from gp130F/F mice. Representative confocal immunofluorescence

photomicrographs of cells stained for (A) cleaved caspase-3 (green) and podoplanin (red, ATI

cell marker) either alone or together, or (B) cleaved caspase-3 (green) and CD31 (red,

endothelial cell marker) either alone or together, in cross-sections of lungs from 6mo gp130F/F

(F/F) mice. In both (A) and (B), sections are also stained with the nuclear marker 4',6-

diamidino-2-phenylindole (DAPI; blue) as indicated. White circles indicate examples of (A)

podoplanin-positive cells (cell membrane) and (B) CD31-positive cells (cell junction in cell

membrane) with cleaved caspase-3 (cytoplasm), while green circles (A and B) indicate

examples of cleaved caspase-3 alone cells. Scale bars = 40μm. The graphs depict stereological

quantification of dual labelled caspase-3/podoplanin- or caspase-3/CD31-positive cells in the

lungs of 6mo mice of the indicated genotypes. Data are expressed as the mean ± SEM. n = 5

mice per genotype.

8

Figure E3: Therapeutic and genetic blockade of IL-6 trans-signalling in CS-exposed wild-

type mice suppresses expression of inflammatory mediators. (A) Q-PCR expression

analyses of the indicated genes were performed on lung cDNA, and (B) ELISAs for the

indicated proteins were performed on lung lysates, from 1 month old gp130+/+:sgp130Fctg/tg

(+/+:tg) mice with (+) or without (-) cigarette smoke (CS) exposure. (C) Q-PCR expression

analyses of inflammatory mediators, Cxcl2, Tnfa and Ccl2 were performed on lung cDNA from

gp130+/+ (+/+) mice without CS (+/+ -CS), with CS (+/+ +CS), or concurrently treated with

sgp130Fc and CS (+/+ Fc+CS), as well as +/+:tg mice with/without CS exposure. Data are

expressed for n = 3 mice per genotype. *P< 0.05 and ** P< 0.01.

Figure E4: Therapeutic and genetic blockade of IL-6TS in gp130F/F mice suppresses

mTORC1 pathway activation in alveolar epithelial type II (ATII) cells. (A and B)

Representative confocal immunofluorescence photomicrographs of cells positive for either P-

rpS6 alone (red, A and B), SPC alone (green, A), CD45 alone (green, B) or both P-rpS6 and

SPC (yellow, white circles, A) in cross-sections of lungs from 6mo gp130F/F (F/F) mice. Scale

bars = 10μm. (C) Immunoblotting with indicated antibodies on whole lung lysates from 6mo

F/F and F/F:Il6-/- mice. Each lane represents tissue from an individual mouse. Densitometric

quantification of P-mTOR (Ser2448) protein levels per genotype was performed and

normalized against Actin protein levels present in each sample. Data are presented as the mean

expression ± SEM from n = 5 mice per genotype. *P< 0.05.

Figure E5: Activation of the IL-6TS/mTORC1 axis in cultured gp130F/F primary alveolar

type II (ATII) epithelial cells stimulated with the IL-6TS agonist Hyper-IL-6. (A)

Immunoblotting with indicated antibodies on lysates from cultured gp130F/F (F/F) ATII cells

following stimulation with Hyper-IL-6 (10ng/ml) over the indicated time points. (B)

9

Representative histograms of flow cytometric analyses of Annexin-V and 7AAD staining

showing percentages of Annexin-V-positive/7AAD-negative apoptotic ATII cells from wild-

type (+/+) and F/F mice.

Figure E6: Blockade of mTORC1 using rapamycin prevents alveolar cell death in

gp130F/F mice with hyper-activated IL-6TS/mTORC1 axis. (A and B) Representative

photomicrographs of P-rpS6-positive cells in cross-sections of lungs from (A) 6mo gp130+/+

(+/+), gp130+/+:sgp130Fctg/tg (+/+:tg), F/F and F/F:tg mice, and (B) 3mo +/+ and F/F mice, and

F/F mice treated with sgp130Fc inhibitor for 12 weeks (F/F + Fc). (C and D) Representative

photomicrographs showing (C) TUNEL-positive cells and (D) P-rpS6-positive cells, in cross-

sections of lungs from gp130F/F (F/F) mice aged 6 months, and 3 month old F/F mice treated

with rapamycin for 12 weeks (F/F + Rapa). Scale bars = 100μm.

Figure E7: Hyper-activation of the IL-6TS/mTORC1 pathway correlates with

augmented cellular apoptosis in the lungs of CS-exposed wild-type mice. (A) Stereological

quantification of the number of P-rpS6-positive cells, and corresponding representative

photomicrographs showing positive cells in a cross-section of lungs from +/+ -CS, +/+ +CS,

and +/+ mice concurrently treated with CS and sgp130Fc (+/+ Fc+CS), as well as

gp130+/+:sgp130Fctg/tg (+/+:tg) mice with or without CS exposure. (B) Stereological

quantification of the number of P-rpS6-positive cells, and corresponding representative

photomicrographs showing positive cells in a cross-section of lungs from +/+ mice

concurrently treated with CS and either vehicle (Veh) or rapamycin (Rapa). (C) Stereological

quantification of the number of TUNEL-positive cells, and corresponding representative

photomicrographs showing positive cells in a cross-section of lungs from +/+ mice

concurrently treated with CS and either Veh or Rapa. Data are expressed as mean ± SEM. n =

10

4 mice per genotype. Scale bars = 100μm. *P< 0.05, **P< 0.01 and ***P< 0.001. (D)

Representative photomicrographs showing P-rpS6-positive cells in cross-sections of the lungs

from gp130+/+ mice (+/+) aged 1 month treated with or without sgp130Fc inhibitor or CS for

12 weeks.

+/+ F/F +/+:tg F/F:tg F/F + sgp130Fc

F/F + Rapa

Vv (par/lung) (%)

87.9(±1.3)

93.9(±2.8)

88.3(±1.7)

91.2(±4.1)

91.9(±1.8)

93.2(±3.3)

Vv (airsp/par) (%)

68.5(±1.0)

80.3###

(±0.6)71.6****(±0.9)

69.4****(±1.5)

71.4****(±0.9)

71.0***(±1.3)

V(airsp/lung) (cm3)

59.6(±2.6)

87.9###

(±2.6)41.6****(±1.6)

55.7****(±5.1)

54.5****(±4.8)

52.3****(±4.4)

Vv (sep/par) (%)

23.2(±1.5)

14.9###

(±0.8)23.9****(±0.9)

23.05***(±0.9)

24.3****(±0.8)

24.0***(±1.0)

V (sep/lung) (cm3)

20.0(±1.0)

16.4#

(±1.1)18.9

(±0.6)18.3

(±1.2)18.5

(±1.5)17.6

(±0.7)

Sv (sep/par) (1/cm)

651(±39)

567#

(±26)623*(±48)

634*(±25)

624*(±21)

639*(±23)

S (sep/lung) (cm2)

538(±28)

583(±34)

501(±19)

511(±6)

499(±56)

535(±38)

Table E1: Comparative stereological analyses of lungs from the indicated mice. Data areexpressed as the mean ± SEM. n = 5 mice per genotype per age group. #P < 0.05 and ###P <0.001 versus age-matched +/+ mice. *P < 0.05, ***P < 0.001 and ****P < 0.0001 versus age-matched F/F mice. Vv = volume fraction; par = parenchyma; air = air space; sep = septal tissue;Sv = surface density; S = surface area.

Table E2: Clinical characteristics of the patients. Data are expressed as the mean ± SEM.FEV1 denotes forced expiratory volume in one second.

Characteristics No emphysema (n=10)

Moderate emphysema (n=20)

Sex (no. of patients)Male

Female55

128

Smoking historyPack years 10 ± 5 42 ± 9Age (years) 69 ± 5 71 ± 2Diagnosis Lung cancer (tissues were

obtained from non-cancer area)

Lung cancer (tissues were obtained from non-cancer

area)FEV1 (% of predicted post)-

COPD status 110.1 ± 4.0 88.5 ± 6.3

TLCO (gas transfer, % predicted post)-emphysema

status

92.8 ± 4.9 51.1 ± 2.0

0

20

40

60

+/+ +/+: tg

F/F F/F: tg

* **

µm

C

A

B

0

5

10

15

00.51.01.52.0

2.5

0

5

10

15

20sIL-6R sgp130IL-6

pg/m

l

pg/m

l

pg/m

l

* *

SAAng

/ml

F/F F/F:tg

Serum

ND ND+/+ F/F +/+ F/F +/+ F/F

0

200

400

600

800

1000

F/F F/F:tg

Lung

Figure E1

Mean linear intercept

A

Figure E2

B

Cleaved Casp-3 + Podoplanin Cleaved Casp-3

Podoplanin Dapi

0

2

4

6

+/+ +/+: tg

F/F F/F: tg

Cleaved Casp-3+/Podoplanin+ cells/

20 fields

Cleaved Casp-3 + CD31 Cleaved Casp-3

CD31 Dapi

0

1

2

3

4

+/+ +/+: tg

F/F F/F: tg

Cleaved Casp-3+/CD31+

cells/20 fields

A

+CS

+/+:tg

0

10

20

30

40

50 ** Ccl2

-CS +CS Fc+CS

+/+

-CS +CS+/+:tg

Rel

ativ

e Ex

pres

sion

-CS +CS Fc+CS

+/+

-CS

C

Rel

ativ

e Ex

pres

sion

Tnfa

0

5

10

15

20 ** *

-CS +CS Fc+CS+/+

-CS +CS+/+:tg

Rel

ativ

e Ex

pres

sion

0

1

2

3

4 sgp130

ng/m

l

-CS +CS

+/+:tg

0

0.2

0.4

0.6

0.8

1.0Il6

-CS +CS

+/+:tg

Rel

ativ

e Ex

pres

sion

10

15

-CS +CS

+/+:tg

sIL-6R

0

5pg/m

l

Cxcl2

0

50

100

150 **

**

***

0

0.2

0.4

0.6

0.8

1.0Adam17

-CS +CS

+/+:tg

B

Figure E3

Rel

ativ

e Ex

pres

sion

Figure E4

A

C F/F F/F:Il6-/-

P-mTOR (ser2448)

mTOR

ActinF/F F/F:Il6-/-

Rat

io to

Act

in

CD45+ P-rpS6

CD45 P-rpS6

SPC+ P-rpS6

SPC P-rpS6

0

0.5

1.0

1.5

P-mTOR (ser2448)

*

B

F/F

F/F

F/F

F/F

Figure E5

F/F

Hyper-IL-6 (hours)

0 0.5 1 3

Actin

S6

P-rpS6

A

B

Cou

nt

Annexin-V-positive/7AAD-negative

F/F+/+

35.2% 45.3%

C F/F + RapaF/F

F/F + RapaF/F

Figure E6

TUNEL

P-rpS6

+/+ F/F F/F + Fc

P-rpS6

P-rpS6

+/+ +/+:tg F/F:tgF/FA

B

D

A

P-rp

S6+

cells

/20

field

s

+/+ (-CS) +/+ (+CS) +/+ (Fc +CS)

+/+:tg (-CS) +/+:tg (+CS)

-CS0

1020304050

+CS Fc+CS

+/+

-CS +CS

+/+:tg

* *

+/+ (Veh +CS) +/+ (Rapa +CS)

Rapa

+/+ +CSVeh

0

20

40

60 ***

P-rp

S6+

cellS

/20

field

s

+/+ +CS

0

5

10

15

20

Veh Rapa

**+/+ (Veh +CS) +/+ (Rapa +CS)

TUN

EL+

cells

/20

field

s

B

C

Figure E7

+/+ (-CS) +/+ (Fc+CS)+/+ (+CS)

P-rpS6

D