Adverse outcome pathways (AOPs) have been developed as a risk assessment tool for regulatory applications. These AOPs describe a logical mechanistic sequence of events, starting with a Molecular Initiating Event (MIE), and ultimately leading to a disease outcome via a series of Key Events (KE). The AOP framework provides a system to make predictions and assessments while reducing the need for in vivo assessment. In the absence of epidemiological evidence, assessment of the health effects of a product, chemical or therapy on the progression of atherosclerosis would necessitate long-term animal exposure studies such as the use of the Apolipoprotein E deficient mouse. We followed Organisation for Economic Co-operation and Development (OECD) guidelines to formulate and propose an AOP for atherosclerotic plaque progression, collating the evidence by which cigarette smoke-induced oxidative stress forms a MIE. The downstream pathway includes multiple KEs including the upregulation of proinflammatory mediators, nitric oxide depletion, and endothelial dysfunction. Alterations in these KEs can lead to plaque formation and progression in cardiovascular disease and increase the risk of morbidity and mortality. Identifying preclinical endpoints and clinical biomarkers associated with these KEs provides a framework for in vitro and clinical data, supporting a mechanistic narrative for regulatory assessment. The application of this pathway provides a powerful alternative to animal models through developing preclinical assays and biomarkers for the assessment of atherosclerosis progression risk.

‘Modern’ oral tobacco-free nicotine pouches (NPs) are a nicotine containing product similar in appearance and concept to Swedish snus. A three-step approach was taken to analyse the biological effects of NPs and snus extracts in vitro. ToxTracker was used to screen for biomarkers for oxidative stress, cell stress, protein damage and DNA damage. Cytotoxicity, mutagenicity, and genotoxicity were assessed in the following respective assays: Neutral Red Uptake (NRU), Ames and Mouse Lymphoma Assay (MLA). Targeted analysis of phosphorylation signalling and inflammatory markers under non-toxic conditions was used to investigate any potential signalling pathways or inflammatory response. A reference snus (CRP1.1) and four NPs with various flavours and nicotine strengths were assessed. Test article extracts was generated by incubating one pouch in 20 mL of media (specific to each assay) with the inclusion of the pouch material. NP extracts did not induce any cytotoxicity or mutagenic response, genotoxic response was minimal and limited signalling or inflammatory markers were induced. In contrast, CRP1.1 induced a positive response in four toxicological endpoints in the absence of S9: Srxn1 (oxidative stress), Btg2 (cell stress), Ddit3 (protein damage) and Rtkn (DNA damage), and three endpoints in presence of S9: Srxn1, Ddit3 and Rtkn. CRP1.1 was genotoxic when assessed in MLA and activated signalling pathways involved in proliferation and cellular stress and specifically induced phosphorylation of c-JUN, CREB1, p53, p38 MAPK and to a lesser extent AKT1S1, GSK3α/β, ERK1/2 and RSK1 in a dose-dependent manner. CRP 1.1 extracts resulted in the release of several inflammatory mediators including cytokines IL-1α, IL5, IL6, IL8, IL-1RA, MIF and TNF-β, receptor IL-2RA, and growth factors FGF-basic, VEGF and M-CSF. In conclusion these assays contribute to the weight of evidence assessment of the potential comparative health risks of NPs and snus.

Consumer demands and innovation have led to an increasingly diverse range of nicotine delivery systems, driven by a desire to reduce risk associated with traditional combustible cigarettes. This speed of change provides a mandate for rapid new product assessment. We have used the validated technology ToxTracker®, to assess biomarkers of DNA damage, protein misfolding, oxidative and cellular stress, across the categories of cigarette (1R6F), tobacco heating product (THP 1.4) and electronic cigarette (ePen 3). In addition, we compared the commonly used test matrices for tobacco and nicotine products; whole aerosol aqueous extracts (AqE) and gas vapour phase (GVP), determining their suitability across the product categories. We demonstrated a significant reduction in oxidative stress and cytotoxicity for THP 1.4 over cigarette, further reduced for ePen 3, when assessed by both dilution and nicotine dosimetry. We also identified that while the extraction matrices AqE and GVP from combustible products were equivalent in the induced responses, this was not true of the other category examples, moreover THP 1.4 GVP demonstrates a >50 % reduction in both toxicity and cytotoxicity endpoints over AqE. This indicates that unlike cigarette, the active components or toxicants for THP and electronic cigarette are associated with the aerosol fraction of these categories.

Goblet cell hyperplasia and overproduction of airway mucin are characteristic features of the lung epithelium of smokers and COPD patients. Tobacco heating products (THPs) are a potentially less risky alternative to combustible cigarettes, and through continued use solus THPs may reduce smoking-related disease risk. Using the MucilAir™ in vitro lung model, a 6-week feasibility study was conducted investigating the effect of repeated cigarette smoke (1R6F), THP aerosol and air exposure. Tissues were exposed to nicotine-matched whole aerosol doses 3 times/week. Endpoints assessed were dosimetry, tight-junction integrity, cilia beat frequency (CBF) and active area (AA), cytokine secretion and airway mucin MUC5AC expression. Comparison of incubator and air exposed controls indicated exposures did not have a significant effect on the transepithelial electrical resistance (TEER), CBF and AA of the tissues. Cytokine secretion indicated clear differences in secretion patterns in response to 1R6F and THP exposure. 1R6F exposure resulted in a significant decrease in the TEER and AA (p=0.000 and p=0.000, respectively), and an increase in MUC5AC positive cells (p=0.002). Repeated THP exposure did not result in a significant change in MUC5AC positive cells. This study demonstrates repeated cigarette smoke whole aerosol exposure can induce these morphological changes in vitro.

Human rhinoviruses (HRVs) express 2 cysteine proteases, 2A and 3C, that are responsible for viral polyprotein processing. Both proteases also suppress host gene expression by inhibiting mRNA transcription, nuclear export and cap-dependent translation. However, the relative contribution that each makes in achieving this goal remains unclear. In this study, we have compared both the combined and individual ability of the two proteases to shut down cellular gene expression using a novel dynamic reporter system. Our findings show that 2A inhibits host gene expression much more rapidly than 3C. By comparing the activities of a representative set of proteases from the three different HRV species, we also find variation in the speed at which host gene expression is suppressed. Our work highlights the key role that 2A plays in early suppression of the infected host cell response and shows that this can be influenced by natural variation in the activity of this enzyme.

Engineering tissue structures that mimic those found in vivo remains a challenge for modern biology. We demonstrate a new technique for engineering composite structures of cells comprising layers of heterogeneous cell types. An acoustofluidic bioreactor is used to assemble epithelial cells into a sheet-like structure. On transferring these cell sheets to a confluent layer of fibroblasts, the epithelial cells cover the fibroblast surface by collective migration maintaining distinct epithelial and fibroblast cell layers. The collective behaviour of the epithelium is dependent on the formation of cell-cell junctions during levitation and contrasts with the behaviour of mono-dispersed epithelial cells where cell-matrix interactions dominate and hinder formation of discrete cell layers. The multilayered tissue model is shown to form a polarised epithelial barrier and respond to apical challenge. The method is useful for engineering a wide range of layered tissue types and mechanistic studies on collective cell migration.

DNA methylation changes in pulmonary fibrosis is a field of study which is increasingly growing in relevance. The DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine (5-aza-DC) has been shown to suppress transforming growth factor-beta-1 (TGFβ1) induced fibrotic changes. To better understand these changes, we studied the anti-fibrotic effect of 5-aza-2’-deoxycytidine on novel 3D multicellular foci derived from individual human lung fibroblast cells. Primary lung fibroblasts were derived from explant tissue of patients who did not have lung disease. These were then grown for a total of six weeks in optimal conditions to promote 3D multicellular foci formation. The multicellular foci were treated with 2.5ng/mL TGFβ1 for the final 5 weeks, and with 1µM 5-aza-DC for the final two weeks. Gross mechanical stiffness of the resulting foci was assessed by parallel plate compression testing. Alpha smooth muscle actin (αSMA) and collagen mRNA expression were assessed by RTqPCR. mRNA expression of collagen cross-linking enzymes, lysyl oxidase (LOX) and lysyl oxidase-like-2 (LOXL2), were also assessed by RTqPCR. Treatment of lung fibroblast-derived 3D foci with co-treatment of 2.5ng/mL TGFβ1 and 1µM 5-aza-DC reduced gross mechanical stiffness 3.5-fold compared to treatment with TGFβ1 alone (p<0.01). The same treatment reduced αSMA mRNA levels by 79.5% (p=0.011). Expression of LOX mRNA was reduced 2.96-fold and LOXL2 mRNA was reduced 4.76-fold (p=0.01). 5-aza-DC ameliorates both the fibrotic response and collagen cross-linking response of human lung fibroblasts to TGFβ1 cultured in this long-term cell culture model.

Accumulation of advanced glycation end-products (AGEs) in biological tissues occurs as a consequence of normal ageing and pathology. Most biological tissues are composed of considerable amounts of collagen, with collagen fibrils being the most abundant form. Collagen fibrils are the smallest discernible structural elements of load-bearing tissues and as such, they are of high biomechanical importance. The low turnover of collagen cause AGEs to accumulate within the collagen fibrils with normal ageing as well as in pathologies. We hypothesized that collagen fibrils bearing AGEs have altered hydration and mechanical properties. To this end, we employed atomic force and Brillouin light scattering microscopy to measure the extent of hydration as well as the transverse elastic properties of collagen fibrils treated with ribose. We find that hydration is different in collagen fibrils bearing AGEs and this is directly related to their mechanical properties. Collagen fibrils treated with ribose showed increased hydration levels and decreased transverse stiffness compared to controlled samples. Our results show that BLS and AFM yield complementary evidence on the effect of hydration on the nanomechanical properties of collagen fibrils.

Sarcoidosis is a multisystem disease of unknown aetiology. Up to 90% of sarcoidosis patients have lung involvement including pulmonary fibrosis. This leads to impaired quality of life and sometimes mortality. To better understand the nature of the lung matrix we generated novel 3D multicellular foci from individual patient cells. Primary lung fibroblasts were isolated from explant tissue from patients with sarcoidosis. These were then grown in optimal conditions to promote 3D multicellular foci formation for a total of six weeks, treated +/- transforming growth factor-beta-1 (TGFβ1). Alpha smooth muscle actin (aSMA) and collagen mRNA expression were assess­ed by RTqPCR and gross mechanical stiffness of the resulting multicellular foci was assessed by parallel plate compression testing. Total collagen was assessed by hydroxyproline assay (ELISA), pyridinoline (PYD) concentrations were measured by ELISA and LC-MS to assess collagen cross-linking. Treatment of sarcoidosis fibroblasts with 2.5ng/mL TGFβ1 caused a 6-fold increase in collagen-1 (p<0.001) and a doubling of aSMA (p<0.05) mRNA. Total collagen protein increased 2.6-fold following TGFb1 treatment (p<0.001), this was associated with a significant increase in gross mechanical stiffness (p<0.005). TGFβ1 increases fibrotic markers in sarcoidosis fibroblasts cultured in this long-term cell culture model.

Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.

CX3CL1 has been implicated in allergen-induced airway CD4 + T-lymphocyte recruitment in asthma. As epidemiological evidence supports a viral infection–allergen synergy in asthma exacerbations, we postulated that rhinovirus (RV) infection in the presence of allergen augments epithelial CX3CL1 release. Fully differentiated primary bronchial epithelial cultures were pretreated apically with house dust mite (HDM) extract and infected with rhinovirus-16 (RV16). CX3CL1 was measured by enzyme-linked immunosorbent assay and western blotting, and shedding mechanisms assessed using inhibitors, protease-activated receptor-2 (PAR-2) agonist, and recombinant CX3CL1-expressing HEK293T cells. Basolateral CX3CL1 release was unaffected by HDM but stimulated by RV16; inhibition by fluticasone or GM6001 implicated nuclear factor-κB and ADAM (A Disintegrin and Metalloproteinase) sheddases. Conversely, apical CX3CL1 shedding was stimulated by HDM and augmented by RV16. Although fluticasone or GM6001 reduced RV16+HDM-induced apical CX3CL1 release, heat inactivation or cysteine protease inhibition completely blocked CX3CL1 shedding. The HDM effect was via enzymatic cleavage of CX3CL1, not PAR-2 activation, yielding a product mitogenic for smooth muscle cells. Extracts of Alternaria fungus caused similar CX3CL1 shedding. We have identified a novel mechanism whereby allergenic proteases cleave CX3CL1 from the apical epithelial surface to yield a biologically active product. RV16 infection augmented HDM-induced CX3CL1 shedding—this may contribute to synergy between allergen exposure and RV infection in triggering asthma exacerbations and airway remodeling.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that usually affects elderly people. It has a poor prognosis and there are limited therapies. Since epigenetic alterations are associated with IPF, histone deacetylase (HDAC) inhibitors offer a novel therapeutic strategy to address the unmet medical need. This study investigated the potential of romidepsin, an FDA-approved HDAC inhibitor, as an anti-fibrotic treatment and evaluated biomarkers of target engagement that may have utility in future clinical trials. The anti-fibrotic effects of romidepsin were evaluated both in vitro and in vivo together with any harmful effect on alveolar type II cells (ATII). Bronchoalveolar lavage fluid (BALF) from IPF or control donors was analyzed for the presence of lysyl oxidase (LOX). In parallel with an increase in histone acetylation, romidepsin potently inhibited fibroblast proliferation, myofibroblast differentiation and LOX expression. ATII cell numbers and their lamellar bodies were unaffected. In vivo, romidepsin inhibited bleomycin-induced pulmonary fibrosis in association with suppression of LOX expression. LOX was significantly elevated in BALF of IPF patients compared to controls. These data show the anti-fibrotic effects of romidepsin, supporting its potential use as novel treatment for IPF with LOX as a companion biomarker for evaluation of early on-target effects.

Aims: To (i) investigate the effects of Romidepsin in vitro using fibroblasts and alveolar cells (ATII); (ii) in vivo in Bleomycin treated mice; and (iii) to identify biomarkers in order to monitor response to therapy in a future clinical trial. Background: Idiopathic pulmonary fibrosis (IPF) is a complex chronic fibroproliferative disease of unknown aetiology and with limited therapeutic options.Numerous 9single pathway9 agents have failed to show efficacy in IPF clinical trial. By targeting multiple genes and pathways, HDAC inhibitors offer novel therapeutic strategies that could be used alone or in combination with existing agents (Pirfenidone and Nintedanib). Methods: Fibroblast and ATII cell proliferation were determined by cell counting and MTS assay. Myofibroblast differentiation was analysed by western blotting (WB) for α-SMA and Lysyl Oxidase (LOX). The antifibrotic effects of Romidepsin on bleomycin treated mice were assessed by RTqPCR, histology and WB. Broncho-alveolar-lavage (BAL) fluid from IPF patients was analysed by WB. Results: Romidepsin caused a strong inhibition of IPF fibroblast proliferation, myofibroblast differentiation and secretion of LOX. Comparison of the effect of Romidepsin on ATII cells and IPF fibroblasts showed that the ATII cells were significantly less sensitive. Romidepsin reduced bleomycin-induced lung fibrosis in mice and suppressed the expression of LOX. We detected elevated levels of LOX in the BALF of IPF patients. Conclusions: Romidepsin shows strong anti-fibrotic effects without harmful consequences on ATII cells. It is therefore a potential novel anti-fibrotic therapy and LOX may be a potential biomarker of response.

The link between airborne particulate matter (PM) and respiratory disease is well established. Early anecdotal observations were based on industrial cases: it is generally accepted that the first observations were made as early as the Greek and Roman civilizations, then called ‘Potter’s Rot’, now known as silicosis, a form of restrictive lung disease or pneumoconiosis resulting from occupational dust inhalation. Silicosis continues to be a significant problem today, albeit having evolved with the changing working practices of industrious men and women and the development of mechanization. The human race has become increasingly adept, creating ever more ingenious ways of generating dust or PM for workers to inhale. With industrialization came, pneumatic drills and grinding of rock, causing further silicosis, foundries, metal mills and welding for airborne reactive metal particles, causing siderosis (Welder’s lung) and even something apparently as benign as cotton dust in cotton mills causing byssinosis (brown lung disease). As recently as 1990, these still accounted for 167 000 lives lost worldwide. Thankfully, there is now improving recognition of these risks and while cases do still occur, generally, efforts are made to reduce these with legal exposure limits and personal protection equipment. In addition, some of the most harmful sources of PM are being replaced and removed. Who has not seen ‘Danger Asbestos Removal’ signage? So where are we going with this? The simple answer is out of the workplace and into the home. Research has identified burning of waste and low cost oil stoves in the developing world to be a source of lung disease, yet these practices have been largely overlooked historically. It could be purely a lack of literary evidence, reduced toxicity and levels of PM to which they were exposed, or an indiscernible phenotype above the populations’ perceived life expectancy. It is also likely however that the PM exposure was generally uniform and accepted and any effects on susceptible individuals were directly attributed and accepted to be part of the general prognosis of that individual’s morbidity. Hence, while there is long standing evidence of respiratory disease resulting from airborne PM exposure in industrial settings resulting from the extremes of exposure, little focus was placed upon the environment and the home. Things can change, as they did in the United Kingdom with the smog of the late 19th and early to mid-20th century. Typical among these was the great London smog of December 1952, which is suggested to have caused up to 12 000 deaths and left 100 000 sick. The smog data combined with data from the newly formed National Health Service of 1948, demonstrated acutely the correlation between ambient airborne PM and respiratory disease, ultimately leading to the United Kingdom Clean Air Act of 1956. Smog continues to be a significant clinical concern in large cities today. These ambient PM events exemplify their potency to induce acute morbidity and mortality especially in susceptible individuals such as those with underlying respiratory diseases like asthma, chronic obstructive pulmonary disease or respiratory infections. It has been estimated that 3.2 million deaths per year can be attributed directly to the effects of particulate matter inhalation, with most patients suffering from existing respiratory diseases. Asthma is one such disease to which ambient airborne PM has been critically associated with exacerbations and hospital admissions. Currently, there are estimated to be 334 million people with asthma worldwide, these are predominantly distributed in the more economically developed regions, with the numbers affected increasing by roughly 50% percent per decade. The link between particulate matter and asthma exacerbation has led to much research being undertaken, not only into the sources of this PM but also its potency to cause these effects. Typically, most attention has been directed to ≤2.5 μm PM fraction (PM2.5) in regard to airborne pollution. Larger fractions deposit primarily in the throat, upper and larger airways with some small airway distribution, while the PM2.5 fraction is able to penetrate deeper into the lungs’ small airways and alveoli. The sources of ambient PM are highly varied and depend on factors such as geographical location with typical sources being building and industry, tyre road wear, diesel exhaust emissions, combustion, photochemical formation, fungal spores, pollen, desert sand storms and even volcanoes. With 380 000 asthma deaths occurring annually, it seems sensible to look at all possible causes and to identify and limit the exposure of the vulnerable. In this month’s Respirology, Anjali Haikerwal and co-authors look at the health effects of the Australian 2006–2007 wildfires, demonstrating that the measured PM2.5 from such wildfires around the state of Victoria correlated directly with hospital admissions bs_bs_banner

Asthma is characterized by periodic episodes of bronchoconstriction and reversible airway obstruction; these symptoms are attributable to a number of factors including increased mass and reactivity of bronchial smooth muscle and extracellular matrix (ECM) in asthmatic airways. Literature has suggested changes in cell responses and signaling can be elicited via modulation of mechanical stress acting upon them, potentially affecting the microenvironment of the cell. In this study, we hypothesized that mechanical strain directly affects the (myo)fibroblast phenotype in asthma. Therefore, we characterized responses of bronchial fibroblasts, from 6 normal and 11 asthmatic non-smoking volunteers, exposed to cyclical mechanical strain using flexible silastic membranes. Samples were analyzed for proteoglycans, α-smooth muscle actin (αSMA), collagens I and III, matrix metalloproteinase (MMP) 2 & 9 and interleukin-8 (IL-8) by qRT-PCR, Western blot, zymography and ELISA. Mechanical strain caused a decrease in αSMA mRNA but no change in either αSMA protein or proteoglycan expression. In contrast the inflammatory mediator IL-8, MMPs and interstitial collagens were increased at both the transcriptional and protein level. The results demonstrate an adaptive response of bronchial fibroblasts to mechanical strain, irrespective of donor. The adaptation involves cytoskeletal rearrangement, matrix remodelling and inflammatory cytokine release. These results suggest that mechanical strain could contribute to disease progression in asthma by promoting inflammation and remodelling responses.

Development of synthetic surfaces that are highly reproducible and biocompatible for in vitro cell culture offers potential for development of improved models for studies of cellular physiology and pathology. They may also be useful in tissue engineering by removal of the need for biologically-derived components such as extracellular matrix proteins. We synthesised four types of 2-alkyl-2-oxazoline polymers ranging from the hydrophilic poly(2-methyl-2-oxazoline) to the hydrophobic poly(2-n-butyl-2-oxazoline). The polymers were terminated using amine-functionalised glass coverslips, enabling the synthetic procedure to be reproducible and scaleable. The polymer-coated glass slides were tested for biocompatibility using human epithelial (16HBE14o-) and fibroblastic (MRC5) cell lines. Differences in adhesion and motility of the two cell types was observed, with the poly(2-isopropyl-2-oxazoline) polymer equally supporting the growth of both cell types, whereas poly(2-n-butyl-2-oxazoline) showed selectivity for fibroblast growth. In summary, 2-alkyl-2-oxazoline polymers may be a useful tool for building in vitro model cell culture models with preferential adhesion of specific cell types.