Interstitial lung disease ORIGINAL RESEARCH Thorax: first published as 10.1136/thoraxjnl-2019-214000 on 24 July 2020. Downloaded from Inhibition of mast cells: a novel mechanism by which nintedanib may elicit anti- fibrotic effects Catherine Overed- Sayer ,1 Elena Miranda ,2 Rebecca Dunmore,3 Elena Liarte Marin,3 Lorea Beloki,3 Doris Rassl,4 Helen Parfrey ,4 Alan Carruthers,3 Amina Chahboub,3 Sofia Koch,2 Gülin Güler-­Gane,5 Michael Kuziora,6 Arthur Lewis ,2 Lynne Murray ,1 Richard May,3 Deborah Clarke3 ► Additional material is ABSTRact published online only. To view Background Idiopathic pulmonary fibrosis (IPF) is a Key messages please visit the journal online (http:// dx. doi. org/ 10. 1136/ chronic and progressive lung disease which presents a thoraxjnl-2019- 214000). grave prognosis for diagnosed patients. Nintedanib (a What is the key question? triple tyrosine kinase inhibitor) and pirfenidone (unclear ► Do mast cells (MCs) correlate with idiopathic For numbered affiliations see mechanism of action) are the only approved therapies pulmonary fibrosis (IPF) disease activity, and end of article. for IPF, but have limited efficacy. The pathogenic can either nintedanib or pirfenidone modulate mechanisms of this disease are not fully elucidated; MC survival or activation? Correspondence to however, a role for mast cells (MCs) has been postulated. Dr Catherine Overed-­Sayer, What is the bottom line? Objectives The aim of this work was to investigate Regeneration, Research ► MCs are increased in IPF lung and negatively and Early Development, a role for MCs in IPF and to understand whether correlate with baseline lung function (FVC) Respiratory and Immunology, nintedanib or pirfenidone could impact MC function. and positively correlate with the number of BioPharmaceuticals R&D, Methods and results MCs were significantly AstraZeneca, Cambridge, CB21 fibroblast foci. Nintedanib, but not pirfenidone, elevated in human IPF lung and negatively correlated 6GH, United Kingdom; can inhibit human MC survival and activation in with baseline lung function (FVC). Importantly, catherine. overed- sayer@ vitro and MC recruitment in the rat bleomycin astrazeneca. com MCs were positively associated with the number of model. fibroblast foci, which has been linked to increased Received 21 August 2019 Revised 17 April 2020 mortality. Furthermore, MCs were increased in the Why read on? Accepted 1 May 2020 region immediately surrounding the fibroblast foci, and ► Inhibition of MC survival or activation may Published Online First co- culture studies confirmed a role for CM –fibroblast be a novel additional mechanism by which 24 July 2020 crosstalk in fibrosis. Nintedanib but not pirfenidone nintedanib exerts anti- fibrotic effects in inhibited recombinant stem cell factor (SCF)–induced patients with IPF. http://thorax.bmj.com/ MC survival. Further evaluation of nintedanib determined that it also inhibited human fibroblast- mediated MC survival. This was likely via a direct effect on ckit (SCF in an abnormal lung structure referred to as the receptor) since nintedanib blocked SCF-stimulated ckit characteristic ‘honeycombing’ morphology, which phosphorylation, as well as downstream effects on MC is associated with impaired lung function.4 The role proliferation and cytokine release. In addition, nintedanib of structural cells such as fibroblasts in the patho- ablated the increase in lung MCs and impacted high genesis of fibrosis is well accepted; however, less on October 2, 2021 by guest. Protected copyright. tissue density frequency (HDFm) in a rat bleomycin model understood is the contribution of immune cells of lung fibrosis. which are also present in IPF lung.5–9 The mast cell Conclusion Nintedanib inhibits MC survival and (MC) is an innate immune cell well known for its activation and thus provides a novel additional involvement in allergy, asthma and in host defence mechanism by which this drug may exert anti- fibrotic against parasites.10 11 However, MCs are also a key effects in patients with IPF. cell responding to tissue injury and are found very early at the site of the wound.12 On activation, MCs can release an array of mediators, which have been shown to partake in all stages of the wound healing INTRODUCTION process, including inflammation, proliferation and Idiopathic pulmonary fibrosis (IPF) is a chronic, re- modelling.13 Thus, given the dysregulated nature progressive and ultimately fatal lung disease with of repair in IPF, this cell could be a potential patho- unknown aetiology and increasing incidence.1 genic link to this disease. © Author(s) (or their employer(s)) 2020. Re-use While still incompletely understood, the pathogen- MCs have been postulated to be pro-fibrotic in permitted under CC BY-­NC. No esis of IPF is thought to involve recurrent injury IPF, although the data are conflicting and their role commercial re-use . See rights to the alveolar epithelium inducing an aberrant remains controversial. Several studies have shown and permissions. Published wound healing response, characterised by activa- that MCs and their mediators, such as histamine, by BMJ. tion and proliferation of fibroblasts and myofibro- are increased in lung tissue and bronchoalveolar 2 14–20 To cite: Overed-S ayer C, blasts. Formation of the fibroblastic foci, as well lavage from patients with IPF. Mechanisti- Miranda E, Dunmore R, et al. as the deposition of extracellular matrix (ECM) cally, reports have demonstrated that human lung Thorax 2020;75:754–763. are downstream pathological features3 that result fibroblast and MC crosstalk leads to increased MC 754 Overed- Sayer C, et al. Thorax 2020;75:754–763. doi:10.1136/thoraxjnl-2019-214000 Interstitial lung disease activation and survival, as well as fibroblast proliferation.17 the MC populations quantified. Subsequently, the outer edge of Others have suggested that the MC mediators tryptase, chymase the foci was extended by 500 µm and MCs in the surrounding Thorax: first published as 10.1136/thoraxjnl-2019-214000 on 24 July 2020. Downloaded from and histamine induce both lung and skin fibroblast proliferation area were quantified. See online supplementary methods for and collagen production,19 21–23 and Shimbori and colleagues more details. recently demonstrated that stretch- induced degranulating MCs 20 activate the pro- fibrotic cytokine TGF-β1. Andersson and MC protease signature analysis in IPF lung colleagues provide evidence for a switch to the connective tissue Additional lung tissue was obtained from patients with IPF MC subtype expressing both chymase and tryptase proteases 24 (n=10) undergoing lung volume reduction surgery or lung (MCTC) in IPF lung, and this increase in MCTCs in IPF has been 20 transplantation at Temple University Lung Center as part of a confirmed recently. In contrast, Cha and colleagues propose a separate study. Patients gave written consent and the study was protective role for lung MCs since patients with higher numbers 16 approved by the institutional review board at Temple Univer- of these cells had less decline in lung function. sity Lung Center. Normal lung tissue samples (n=9) were Stem cell factor (SCF) is a key survival factor for mast cells, obtained from the National Disease Research Interchange acting through the tyrosine kinase receptor ckit (also known as (NDRI), in accordance with local internal review board ethical CD117 or SCF receptor). Importantly, a phase II clinical trial approval. Microarray analysis was performed using Affymetrix with a ckit inhibitor Imatinib (Gleevec) had no significant effect HG- U133Plus2.0 microarrays. For more information, see online on lung function decline (or mortality), although the effect on supplementary methods. MCs was not determined in this study.25 Recently, two small molecule drugs, pirfenidone and nintedanib, were licensed for IPF following successful phase Cell culture and in vitro assays III clinical trials demonstrating a slowing in lung function Cord blood–derived MCs (CBMCs) were differentiated from + decline.26–28 Pirfenidone is a small molecule whose precise target CD133 cord blood precursors (Lonza). These cells are predom- is unknown, that exerts effects on transforming growth factor inantly of the MCT phenotype (approximately 70%), as deter- (TGF)-β1 signalling and PDGF as well as acting as an antioxi- mined using a similar staining protocol to that used to phenotype dant,29 whereas nintedanib is a broad tyrosine kinase inhibitor the lung tissue MCs. Conditioned media was prepared from with known activity on platelet-derived growth factor (PDGF), CBMCs that had been cultured for 7 days. CBMC lysates were fibroblast growth factor (FGF) and vascular endothelial growth prepared by sonication and freeze thawing. See online supple- factor (VEGF) receptors.30 Thus, it is plausible that nintedanib mentary methods for details. may also have activity against the ckit receptor, which is found on MCs. Notably, the effects of nintedanib or pirfenidone on CBMC survival MC survival and function have not yet been investigated. CBMCs were cultured with recombinant SCF (MedImmune, In this study, we sought to confirm that MCs are increased in see online supplementary methods for details) in the pres- IPF versus non- diseased lung, and compared MC density in other ence or absence of nintedanib, pirfenidone (both Selleckchem. fibrotic lung diseases such as chronic hypersensitivity pneumo- com) or DMSO vehicle (Sigma Aldrich). After 7 days, CBMCs nitis (HP) and non-specific interstitial pneumonia (NSIP). We were stained