Idiopathic pulmonary fibrosis (IPF) is a devastating disease of the lung, which scars the tissue and gradually destroys the organ, ultimately leading to death. It is still unclear what exactly causes this scarring, but it is thought that increasing amounts of proteins in the space surrounding the cells of the lungs, the extracellular matrix, could play a role.

These proteins, including collagen, normally form a ‘scaffold’ to stabilize cells, but if they accumulate uncontrollably, they can render tissues rigid. It has been assumed that these changes are a consequence of the disease. However, recent evidence suggests that the increased stiffness itself could stimulate cells to produce even more extracellular matrix, driving the progression of the disease. A better understanding of what exactly causes the tissue to become gradually stiffer may identify new ways to block the progression of IPF.

Now, Jones et al. compared measurements of the tissue stiffness and the collagen structure taken from samples of patients with IPF. The results showed that the collagen fibres were faulty and had an abnormal shape. This suggests that these problems, rather than an increased amount of collagen, alter the flexibility of the lung tissue.

Jones et al. also found that a specific family of proteins, which helps to connect the collagen fibres, was increased in the tissue of patients with IPF. When these proteins were blocked with a newly developed drug, the collagen structure returned to normal and the stiffness of the tissue decreased. As a consequence, the lung capacity improved.

This suggests that treatment approaches that help to maintain a normal collagen structure, may in future prevent the stiffening of the lung tissue and so limit feed-forward mechanisms that drive progressive IPF. Moreover, it indicates that measurements of the structure of collagen rather than the its total concentration could serve as a more suitable indicator for the disease.

Fibrotic diseases are a major cause of morbidity and mortality worldwide and their prevalence is increasing with an ageing population. Within the lung, idiopathic pulmonary fibrosis (IPF) is considered the prototypic chronic progressive fibrotic disease (Raghu et al., 2011). Treatment options are limited, and with a median survival of less than 3 years from diagnosis, more effective therapies are urgently needed (Richeldi et al., 2017). Whilst the exact mechanisms of progressive lung fibrosis are uncertain, IPF is thought to result from repetitive micro-injuries to the alveolar epithelium promoting fibroblast differentiation into extracellular matrix (ECM)-producing myofibroblasts. Persistent myofibroblast activation results in ECM deposition which eventually destroys normal alveolar architecture and disrupts gas exchange (Kirk et al., 1986).

Excessive deposition of fibrillar collagens is considered synonymous with fibrosis. Fibrillar collagens are a major component of lung ECM that form a scaffold to support tissue architecture and are a primary determinant of tissue stiffness (Senior et al., 1975; White, 2015). During biosynthesis, collagen molecules acquire several post-translational modifications, including lysine hydroxylation and oxidation that are critical to the structure and biological functions of this protein. Oxidative deamination of lysine or hydroxylysine initiates cross-linking reactions that are essential to stabilise the supramolecular assembly of collagen and produce stable collagen fibrils (Shoulders and Raines, 2009; Kadler et al., 1996). The lysyl oxidase (LOX) enzymes are a family of five secreted copper-dependent amine oxidases (LOX and LOX-like (LOXL) 1 to 4) that are responsible for post-translational modification of collagen in the ECM to initiate covalent cross-linking. LOX family members have been implicated as possible therapeutic targets in cancers and fibrosis (Trackman, 2016). The type of LOX/LOXL mediated collagen cross-link is determined by the hydroxylation of telopeptidyl and helical lysine residues in collagen prior to cross-link formation (Yamauchi and Sricholpech, 2012), with increased hydroxylation of telopeptide lysine residues by lysyl hydroxylase 2/procollagen lysine,2-oxoglutarate 5-dioxygenase 2 (LH2/PLOD2) proposed to be a general fibrotic phenomenon causing increased hydroxyallysine derived pyridinoline cross-links (Brinckmann et al., 1999; van der Slot et al., 2003).

Both ECM amount and altered post-translational modifications are postulated to increase matrix stiffness and this stiffening has been proposed to induce self-sustaining mesenchymal cell activation and progressive fibrosis in a positive feedback loop (Wipff et al., 2007; Liu et al., 2010; Zhou et al., 2013; Booth et al., 2012; Chen et al., 2016; Parker et al., 2014; Shi et al., 2011; Liu et al., 2015). Whilst there is a growing understanding of mechanosensitive cellular pathways that are activated by increased ECM stiffness, the specific changes in ECM structure and function that disrupt mechano-homeostasis have not been defined in human fibrosis (Burgstaller et al., 2017; Tschumperlin et al., 2018; Herrera et al., 2018). To investigate this, we performed the first integrated multiscale structure-function analysis of human fibrosis tissue, and then extended our findings into mechanistic studies in vitro and in vivo. We identify that, at the time of diagnosis, in human lung fibrosis tissue altered collagen architecture rather than collagen concentration is a key determinant of abnormal ECM structure-function, and that targeting pathways which dysregulate collagen architecture may restore ECM homeostasis and so prevent persistent mechanosensitive cellular activation and fibrosis progression.

One of the critical issues in the pathobiology of fibrosis is an insufficient understanding of the structure-function relationship of the ECM in human disease. A major goal of this study was to perform an integrated biomechanical and biochemical characterisation of fibrillar collagen and its pathological role in human fibrosis. We demonstrate that dysregulated collagen fibrillogenesis is a key pathway of human lung fibrosis at the time of diagnosis, with structurally and functionally abnormal collagen – rather than collagen content - increasing lung tissue stiffness as a consequence of pathologic pyridinoline cross-linking. To advance our mechanistic understanding of these findings, we employed small molecule LOXL inhibitors and demonstrate that dual inhibition of LOXL2 and LOXL3 is sufficient to prevent accumulation of these pyridinoline cross-links. This normalises the structure and biomechanical properties of fibrillar collagen, and reduces tissue stiffness without the need to significantly disrupt fibril assembly. Our findings suggest that this targeted inhibition reduces the disruption of mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.

A thought provoking finding of our studies is that total collagen concentration, as determined by measurement of hydroxyproline that quantifies all collagen irrespective of whether it is assembled into fibrils, was not significantly increased in IPF tissue. Whilst Selman et al identified an increase in collagen of fibrotic lung biopsy samples (Selman et al., 1986), other studies have reported no significant increase in collagen deposition (Nkyimbeng et al., 2013; Fulmer et al., 1980; Westergren-Thorsson et al., 2017), or have reported increased collagen deposition only in late stage disease at autopsy, but not in diagnostic lung biopsy samples (Kirk et al., 1986). This suggests that, at the time of diagnosis, any increase in collagen is paralleled by changes in other proteins (most likely other ECM and myofibroblastic cellular proteins), but that by late-stage fibrosis there may be a progressive increase in collagen accumulation, perhaps as the fibrotic tissue becomes more acellular and/or the collagen resists degradation. Furthermore, although we identified an increase in stiffness of IPF lung tissue using AFM micro-indentation, this was not related to collagen concentration. Rather, increased mature fibrillar collagen pyridinoline cross-linking secondary to dysregulated post-translational modification was a primary determinant of tissue stiffness.

Fibrillar collagen is a predominant component of the ECM which provides tissues with essential tensile strength by forming a hierarchical assembly of substructures dependent upon their cross-linking: tropocollagen molecules assemble into fibrils and the fibrils further assemble into fibres. Here, we identified that collagen hierarchical assembly is altered at the nanoscale/ultrastructural level in IPF. Collagen fibrils were structurally and functionally abnormal with reduced diameter and increased stiffness, suggesting that at a molecular level they have the potential to influence the microenvironment of individual cells to affect their biomechanical sensing. Covalent cross-links have been proposed to increase fibril stiffness by forming a lateral network of linkages (Bailey, 2001; Miles et al., 2005). In models of artificially cross-linked collagen fibrils, it was observed that by drawing the collagen molecules closer together there was a reduction in water between molecules; as a consequence, the density of collagen fibrils increased and this was proposed to affect their mechanical properties (Bailey, 2001; Miles et al., 2005). Whether a similar effect occurs after enzymatic cross-linking, especially pathological cross-linking involving pyridinoline cross-links, remains to be determined. However, the reduction in fibril diameter and lesser fibril swelling with hydration of IPF fibrils would be consistent with this concept. Whilst direct experimental evidence is required to determine whether collagen swelling is directly related to cross-linking, collagen hydration alone has been shown to significantly influence individual collagen fibril mechanics and the mechanism of molecular slippage and stretching within fibrils (Andriotis et al., 2018; Gautieri et al., 2011; Yang et al., 2008).

Several different mechanisms have been proposed to contribute to alterations in tissue stiffness including LOX/LOXL and transglutaminase 2 (TGM2)-mediated enzymatic cross-linking and non-enzymatic glycation. Here we have focussed on the LOX and LOXL enzymes which play key roles in the process of fibrillar collagen production, with expression being tightly controlled in normal development (Trackman, 2016). Whilst LOXL2 has been proposed to have pathologic roles in cancer and fibrosis (Barker et al.; Barry-Hamilton et al., 2010), and increased LOXL2 expression in IPF stroma has been observed previously, less is understood regarding LOXL3. Previous studies have identified that it is expressed in human IPF lung tissue, has amine oxidase activity for collagens, is induced by fibroblast culture on IPF cell-derived ECM, and is an enhancer and key regulator of integrin signalling in myofibre formation (Kraft-Sheleg et al., 2016; Lee and Kim, 2006; Philp et al., 2018). Recently, both LOXL2 and LOXL3 were identified to be crucial for fibroblast-to-myofibroblast transition in in vitro models of lung fibrosis (Aumiller et al., 2017). In keeping with our finding of increased LOXL expression together with increased LH2 expression in IPF tissue, we identified an increase in mature hydroxyallysine-derived cross-links in IPF lung tissue, consistent with a previous report of an increase in mature DPD cross-links in fibrotic lung tissue (Last et al., 1990). The extent of lysine hydroxylation can vary from 15 to 90% depending on the collagen types and, even within type I collagen, it varies significantly from tissue to tissue and under physiological or pathological conditions (Yamauchi and Sricholpech, 2012). Although it is unknown whether LOXL2 and/or LOXL3 have a substrate preference for hydroxylysine, the shift from a skin (allysine-derived) to a bone-type (hydroxyallysine-derived) of collagen cross-link appears of pathogenetic importance in IPF.

Various approaches have investigated the roles of collagen cross-linking by LOX/LOXL enzymes in fibrosis. Multiple studies have tested BAPN as a broad-spectrum LOX/LOXL inhibitor (Trackman, 2016), although this approach has no therapeutic potential as it does not enable specific enzyme targeting and has undesirable effects on LOX leading to unwanted vascular and skeletal side-effects (Wawzonek et al., 1955). A monoclonal antibody which binds and partially inhibits LOXL2, showed efficacy in the bleomycin mouse model of lung fibrosis (Barry-Hamilton et al., 2010). However, recently, a Phase 2 study in IPF of simtuzumab, the humanised version of this antibody, was terminated due to lack of efficacy (Raghu et al., 2017). Our finding of differential expression of LOXL2, LOXL3 and LOXL4 in IPF tissue suggests that therapeutic targeting of additional LOXL family members is required to inhibit pathologic collagen cross-linking. To investigate this finding, we used dual LOXL2 and LOXL3-selective inhibitors which irreversibly inhibit LOXL2 and LOXL3 but are reversible and more than a hundred times less potent for inhibition of LOX and LOXL1. Characterisation in a novel 3D in vitro model of human fibrosis enabled direct measurement of the influence of LOXL2/LOXL3 inhibition upon pathological changes in collagen structure-function.

A key finding of our study is that approximately 70% of maximal reduction in tissue stiffness in vitro occurred following selective inhibition of only LOXL2/LOXL3 when pyridinoline cross-links were significantly reduced and fibril diameter was normalised, whilst fibril architecture was preserved and only a small increase in protease-sensitive collagen solubility was evident. Together our data suggest that in lung fibrosis reducing pathological collagen cross-linking normalises fibril architecture and is sufficient to reduce tissue stiffness without the need to completely disrupt fibril assembly. While LOXL4 was also increased in IPF, our dose-response findings from the parallel plate compression system suggest that it does not make a significant contribution to tissue stiffness. In comparison with selective LOXL2/LOXL3 inhibition in the current study, previous studies treating tendon constructs from days 14 to 21 with the pan LOX/LOXL inhibitor BAPN showed that collagen fibrils were irregular with widely dispersed diameters (Herchenhan et al., 2015) and we observed a similar effect of BAPN in the current study. Thus, while pan LOX/LOXL inhibition disrupts normal collagen fibrillogenesis, selective LOXL2/LOXL3 inhibition was sufficient to prevent pathological collagen crosslinking and restore fibrillar collagen homeostasis.

Although reduction in hydroxyproline measurements of collagen is recommended as a primary measure of anti-fibrotic efficacy during preclinical animal model testing of putative therapeutics for lung fibrosis (ATS Assembly on Respiratory Cell and Molecular Biology et al., 2017), our data from human tissue obtained at time of diagnosis identifies that effects on collagen fibrillogenesis are more relevant pathogenic mechanisms with abnormal collagen cross-linking and increased tissue stiffness likely to precede any accumulation of collagen in late-stage human pulmonary fibrosis. Consistent with our proposal, in a rat carbon tetrachloride-induced model of liver injury, increased liver stiffness was associated with increased collagen cross-linking and this was proposed to prime the liver to respond quickly to injury via mechanical feed-forward mechanisms (Perepelyuk et al., 2013; Georges et al., 2007).

The relevance of LOXL2/LOXL3-selective inhibition was demonstrated in a TGF-β-driven rat model of lung fibrosis. The model leads to persistent and severe interstitial fibrosis and does not have the confounding robust inflammatory response observed in the commonly studied bleomycin induced mouse model of fibrosis so enabling direct assessment of fibrotic responses (Sime et al., 1997; ATS Assembly on Respiratory Cell and Molecular Biology et al., 2017). Selective targeting of LOXL2 and LOXL3 in this model reduced pyridinoline collagen cross-links, reduced fibrosis, and improved lung function without a significant effect on hydroxyproline content. Whilst this did not completely restore lung function back to normal, there was a significant beneficial effect even in this model of fibrosis which is very strongly driven by TGF-β. An interesting finding was that inhibition of LOXL2 and LOXL3 reduced expression of genes known to influence collagen fibrillogenesis including LH2, collagen V, TGM2, dermatopontin, fibulin, fibrillin, and periostin. As these genes, as well as LOXL2 and LH2 are induced by TGF-β, one possible explanation is that TGF-β plays a causal role in fibrosis by disrupting collagen homeostasis to drive abnormal collagen cross-linking and promoting assembly of stiffer collagen fibrils. This change in stiffness initiates self-sustaining mechanosensory signalling pathways in a feed-forward mechanism to drive fibroblast activation and progressive fibrosis (Herrera et al., 2018; Tschumperlin et al., 2018). Given the important contribution of fibrillar collagen in forming a scaffold that supports tissue architecture, our data identify that normalising the biomechanical properties of fibrillar collagen in lung fibrosis may reset mechanosensitive cellular mechanisms and aid restoration of tissue function more effectively than approaches that promote extensive collagen degradation, since significant loss of ECM structure is itself pathological, as evidenced by the emphysematous lung.

In summary, this integrated study identifies that altered collagen architecture is a key determinant of abnormal ECM structure-function in human lung fibrosis at time of diagnosis. Dual inhibition of LOXL2 and LOXL3 reduces pathological pyridinoline cross-links, normalises collagen morphology, reduces tissue stiffness, and improves lung function. Thus, targeting pathways which dysregulate collagen architecture may restore ECM homeostasis and so prevent persistent mechanosensitive cellular activation and fibrosis progression.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Mark G Jones

   NIHR Southampton Biomedical Research Centre, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   mark.jones@soton.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6308-6014

2. Orestis G Andriotis

   Institute for Lightweight Design and Structural Biomechanics, TU Wien, Getreidemarkt, Austria

   Contribution

   Formal analysis, Investigation, Methodology, Writing—review and editing

   Contributed equally with

   James JW Roberts

   Competing interests

   No competing interests declared

3. James JW Roberts

   Synairgen Research Ltd, Southampton, United Kingdom

   Contribution

   Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Orestis G Andriotis

   Competing interests

   As a current employee of Synairgen Research Ltd, J.J.W.R has share options in the company.

4. Kerry Lunn

   Synairgen Research Ltd, Southampton, United Kingdom

   Contribution

   Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   As a current employee of Synairgen Research Ltd, K.L has share options in the company.

5. Victoria J Tear

   Synairgen Research Ltd, Southampton, United Kingdom

   Contribution

   Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   As a current employee of Synairgen Research Ltd, V.J.T has share options in the company.

6. Lucy Cao

   Pharmaxis Ltd, Frenchs Forest, Australia

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   As a former or current employee of Pharmaxis Pharmaceuticals Ltd, L.C. has an equity stake in the company.

7. Kjetil Ask

   Department of Medicine, Firestone Institute for Respiratory Health, McMaster University and The Research Institute of St. Joe's Hamilton, Hamilton, Canada

   Contribution

   Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

8. David E Smart

   NIHR Southampton Biomedical Research Centre, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom

   Contribution

   Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

9. Alessandra Bonfanti

   Aeronautics, Astronautics and Computational Engineering, Faculty of Engineering and the Environment, University of Southampton, Southampton, United Kingdom

   Contribution

   Formal analysis, Writing—review and editing

   Competing interests

   No competing interests declared

10. Peter Johnson

    1. Department of Chemistry, Faculty of Natural and Environmental Sciences, University of Southampton, Southampton, United Kingdom
    2. Institute for Life Sciences, University of Southampton, Southampton, United Kingdom

    Contribution

    Investigation, Writing—review and editing

    Competing interests

    No competing interests declared

11. Aiman Alzetani

    1. NIHR Southampton Biomedical Research Centre, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom
    2. University Hospital Southampton, Southampton, United Kingdom

    Contribution

    Resources, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

12. Franco Conforti

    NIHR Southampton Biomedical Research Centre, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom

    Contribution

    Resources, Investigation, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

13. Regan Doherty

    Biomedical Imaging Unit, Faculty of Medicine, University of Southampton, Southampton, United Kingdom

    Contribution

    Investigation, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

14. Chester Y Lai

    NIHR Southampton Biomedical Research Centre, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom

    Contribution

    Resources, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

15. Benjamin Johnson

    CRUK and NIHR Experimental Cancer Medicine Centre, Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom

    Contribution

    Resources, Writing—review and editing

    Competing interests

    No competing interests declared

16. Konstantinos N Bourdakos

    1. Department of Chemistry, Faculty of Natural and Environmental Sciences, University of Southampton, Southampton, United Kingdom
    2. Institute for Life Sciences, University of Southampton, Southampton, United Kingdom

    Contribution

    Resources, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

17. Sophie V Fletcher

    1. NIHR Southampton Biomedical Research Centre, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom
    2. University Hospital Southampton, Southampton, United Kingdom

    Contribution

    Resources, Writing—review and editing

    Competing interests

    No competing interests declared

18. Ben G Marshall

    1. NIHR Southampton Biomedical Research Centre, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom
    2. University Hospital Southampton, Southampton, United Kingdom

    Contribution

    Resources, Project administration, Writing—review and editing

    Competing interests

    No competing interests declared

19. Sanjay Jogai

    University Hospital Southampton, Southampton, United Kingdom

    Contribution

    Resources, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

20. Christopher J Brereton

    NIHR Southampton Biomedical Research Centre, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom

    Contribution

    Investigation, Writing—review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-8302-702X

21. Serena J Chee

    1. University Hospital Southampton, Southampton, United Kingdom
    2. CRUK and NIHR Experimental Cancer Medicine Centre, Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom

    Contribution

    Resources, Project administration, Writing—review and editing

    Competing interests

    No competing interests declared

22. Christian H Ottensmeier

    CRUK and NIHR Experimental Cancer Medicine Centre, Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom

    Contribution

    Resources, Project administration, Writing—review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3619-1657

23. Patricia Sime

    Division of Pulmonary and Critical Care Medicine, University of Rochester School of Medicine and Dentistry, Rochester, United States

    Contribution

    Resources, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

24. Jack Gauldie

    Department of Medicine, Firestone Institute for Respiratory Health, McMaster University and The Research Institute of St. Joe's Hamilton, Hamilton, Canada

    Contribution

    Formal analysis, Investigation, Writing—review and editing

    Competing interests

    No competing interests declared

25. Martin Kolb

    Department of Medicine, Firestone Institute for Respiratory Health, McMaster University and The Research Institute of St. Joe's Hamilton, Hamilton, Canada

    Contribution

    Formal analysis, Investigation, Writing—review and editing

    Competing interests

    No competing interests declared

26. Sumeet Mahajan

    1. Department of Chemistry, Faculty of Natural and Environmental Sciences, University of Southampton, Southampton, United Kingdom
    2. Institute for Life Sciences, University of Southampton, Southampton, United Kingdom

    Contribution

    Investigation, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

27. Aurelie Fabre

    Department of Histopathology, St. Vincent's University Hospital & UCD School of Medicine, University College Dublin, Dublin, Ireland

    Contribution

    Investigation, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

28. Atul Bhaskar

    Aeronautics, Astronautics and Computational Engineering, Faculty of Engineering and the Environment, University of Southampton, Southampton, United Kingdom

    Contribution

    Formal analysis, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

29. Wolfgang Jarolimek

    Pharmaxis Ltd, Frenchs Forest, Australia

    Contribution

    Resources, Investigation, Methodology, Writing—review and editing

    Competing interests

    As a current employee of Pharmaxis Pharmaceuticals Ltd, W.J. has an equity stake in the company.

30. Luca Richeldi

    1. NIHR Southampton Biomedical Research Centre, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom
    2. Università Cattolica del Sacro Cuore, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy

    Contribution

    Conceptualization, Formal analysis, Writing—review and editing

    Competing interests

    No competing interests declared

31. Katherine MA O'Reilly

    1. Mater Misericordiae University Hospital, Dublin, Ireland
    2. School of Medicine and Medical Science, University College Dublin, Dublin, Ireland

    Contribution

    Conceptualization, Formal analysis, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

32. Phillip D Monk

    Synairgen Research Ltd, Southampton, United Kingdom

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Investigation, Methodology, Project administration, Writing—review and editing

    Contributed equally with

    Philipp J Thurner and Donna E Davies

    Competing interests

    As a current employee of Synairgen Research Ltd, P.D.M has share options and is a share-holder in the company.

33. Philipp J Thurner

    Institute for Lightweight Design and Structural Biomechanics, TU Wien, Getreidemarkt, Austria

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—review and editing

    Contributed equally with

    Phillip D Monk and Donna E Davies

    Competing interests

    No competing interests declared

34. Donna E Davies

    1. NIHR Southampton Biomedical Research Centre, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom
    2. Institute for Life Sciences, University of Southampton, Southampton, United Kingdom

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

    Contributed equally with

    Phillip D Monk and Philipp J Thurner

    For correspondence

    d.e.davies@soton.ac.uk

    Competing interests

    D.E.D is co-founder of, share-holder in and consultant to Synairgen Research Ltd.

    "This ORCID iD identifies the author of this article:" 0000-0002-5117-2991

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