Carmel Stock

Mucin 5B promoter polymorphism is associated with idiopathic pulmonary fibrosis but not with development of lung fibrosis in systemic sclerosis or sarcoidosis

Background A polymorphism (rs35705950) 3 kb upstream of MUC5B, the gene encoding Mucin 5 subtype B, has been shown to be associated with familial and sporadic idiopathic pulmonary fibrosis (IPF). We set out to verify whether this variant is also a risk factor for fibrotic lung disease in other settings and to confirm the published findings in a UK Caucasian IPF population. Methods Caucasian UK healthy controls (n=416) and patients with IPF (n=110), sarcoidosis (n=180) and systemic sclerosis (SSc) (n=440) were genotyped to test for association. The SSc and sarcoidosis cohorts were subdivided according to the presence or absence of fibrotic lung disease. To assess correlation with disease progression, time to decline in forced vital capacity and/or lung carbon monoxide transfer factor was used in the IPF and SSc groups, while a persistent decline at 4 years since baseline was evaluated in patients with sarcoidosis. Results A significant association of the MUC5B promoter single nucleotide polymorphism with IPF (p=2.04×10–17; OR 4.90, 95% CI 3.42 to 7.03) was confirmed in this UK population. The MUC5B variant was not a risk factor for lung fibrosis in patients with SSc or sarcoidosis and did not predict more rapidly progressive lung disease in any of the groups. Rather, a trend for a longer time to decline in forced vital capacity was observed in patients with IPF. Conclusions We confirm the MUC5B variant association with IPF. We did not observe an association with lung fibrosis in the context of SSc or sarcoidosis, potentially highlighting fundamental differences in genetic susceptibility, although the limited subgroup numbers do not allow a definitive exclusion of an association.

Host–Microbial Interactions in Idiopathic Pulmonary Fibrosis

Rationale: Changes in the respiratory microbiome are associated with disease progression in idiopathic pulmonary fibrosis (IPF). The role of the host response to the respiratory microbiome remains unknown. Objectives: To explore the host‐microbial interactions in IPF. Methods: Sixty patients diagnosed with IPF were prospectively enrolled together with 20 matched control subjects. Subjects underwent bronchoalveolar lavage (BAL), and peripheral whole blood was collected into PAXgene tubes for all subjects at baseline. For subjects with IPF, additional samples were taken at 1, 3, and 6 months and (if alive) 1 year. Gene expression profiles were generated using Affymetrix Human Gene 1.1 ST arrays. Measurements and Main Results: By network analysis of gene expression data, we identified two gene modules that strongly associated with a diagnosis of IPF, BAL bacterial burden (determined by 16S quantitative polymerase chain reaction), and specific microbial operational taxonomic units, as well as with lavage and peripheral blood neutrophilia. Genes within these modules that are involved in the host defense response include NLRC4, PGLYRP1, MMP9, and DEFA4. The modules also contain two genes encoding specific antimicrobial peptides (SLPI and CAMP). Many of these particular transcripts were associated with survival and showed longitudinal overexpression in subjects experiencing disease progression, further strengthening the relationship of the transcripts with disease. Conclusions: Integrated analysis of the host transcriptome and microbial signatures demonstrated an apparent host response to the presence of an altered or more abundant microbiome. These responses remained elevated in longitudinal follow‐up, suggesting that the bacterial communities of the lower airways may act as persistent stimuli for repetitive alveolar injury in IPF.

Microarray profiling reveals suppressed interferon stimulated gene program in fibroblasts from scleroderma-associated interstitial lung disease

BackgroundInterstitial lung disease is a major cause of morbidity and mortality in systemic sclerosis (SSc), with insufficiently effective treatment options. Progression of pulmonary fibrosis involves expanding populations of fibroblasts, and the accumulation of extracellular matrix proteins. Characterisation of SSc lung fibroblast gene expression profiles underlying the fibrotic cell phenotype could enable a better understanding of the processes leading to the progressive build-up of scar tissue in the lungs. In this study we evaluate the transcriptomes of fibroblasts isolated from SSc lung biopsies at the time of diagnosis, compared with those from control lungs.MethodsWe used Affymetrix oligonucleotide microarrays to compare the gene expression profile of pulmonary fibroblasts cultured from 8 patients with pulmonary fibrosis associated with SSc (SSc-ILD), with those from control lung tissue peripheral to resected cancer (n=10). Fibroblast cultures from 3 patients with idiopathic pulmonary fibrosis (IPF) were included as a further comparison. Genes differentially expressed were identified using two separate analysis programs following a set of pre-determined criteria: only genes significant in both analyses were considered. Microarray expression data was verified by qRT-PCR and/or western blot analysis.ResultsA total of 843 genes were identified as differentially expressed in pulmonary fibroblasts from SSc-ILD and/or IPF compared to control lung, with a large overlap in the expression profiles of both diseases. We observed increased expression of a TGF-β response signature including fibrosis associated genes and myofibroblast markers, with marked heterogeneity across samples. Strongly suppressed expression of interferon stimulated genes, including antiviral, chemokine, and MHC class 1 genes, was uniformly observed in fibrotic fibroblasts. This expression profile includes key regulators and mediators of the interferon response, such as STAT1, and CXCL10, and was also independent of disease group.ConclusionsThis study identified a strongly suppressed interferon-stimulated gene program in fibroblasts from fibrotic lung. The data suggests that the repressed expression of interferon-stimulated genes may underpin critical aspects of the profibrotic fibroblast phenotype, identifying an area in pulmonary fibrosis that requires further investigation.

Mucins MUC5B and MUC5AC in Distal Airways and Honeycomb Spaces: Comparison among Idiopathic Pulmonary Fibrosis/Usual Interstitial Pneumonia, Fibrotic Nonspecific Interstitial Pneumonitis, and Control Lungs

Although the pathogenesis of idiopathic pulmonary fibrosis (IPF) remains elusive (1), one of the most intriguing aspects concerns the possible role of mucins. A strong association has been reported between the promoter polymorphism rs35705950 of MUC5B and the occurrence of familial/sporadic IPF (2–10), as well as with a more benign disease course (10, 11). Overexpression of MUC5B and of the other main airway mucin, MUC5AC, has been described in IPF lungs (12, 13), but the level of expression in other types of pulmonary fibrosis is unknown. In this study, we compare MUC5B and MUC5AC expression among IPF, idiopathic nonspecific interstitial pneumonitis (i-NSIP), systemic sclerosis–associated NSIP (SSc-NSIP), and control lungs. Some of the results of this study have been previously reported in the form of an abstract (14). Surgical lung biopsies from 23 patients with IPF/usual interstitial pneumonia (UIP) (17 men; mean6 SD age, 596 10 yr; 16 ever-smokers), 18 with i-NSIP (10 men; mean, age 466 23 yr; 11 ever-smokers), and 15 with SSc-NSIP (4 men; mean age, 526 11 yr; 11 ever-smokers) and normal lung tissue peripheral to resected cancer from 10 smoker and 10 nonsmoker control subjects (11 men; mean age, 686 14 yr) were selected from the Royal Brompton Hospital pathology archive with ethical approval. No significant differences in FVC, diffusing capacity of the lung for carbon monoxide (DLCO), or composite physiologic index (15) were observed between the different fibrotic patterns with the exception of patients with SSc-NSIP, characterized by a significantly higher DLCO (FVC, 796 22, 796 15, and 786 26% of the predicted value, respectively, in IPF, SSc-NSIP, and i-NSIP; DLCO, 516 9, 586 8, and 486 15% of the predicted value; composite physiologic index, 416 10, 386 7, 476 14). Sequential sections were immunolabeled with MUC5Band MUC5ACspecific antibodies (MUC5AC, Clone 45M1; Biocare Ltd., Concord, CA; and MUC5B, clone sc-20119, Santa Cruz Biotechnology, Dallas, TX; 1:100 dilution for both). Distal airways were defined as airways with no cartilage support or glandular elements, surrounded by smooth muscle bands and characterized by an undulating epithelium. Honeycomb cysts were defined as mucuscontaining areas with a less-wrinkled epithelium compared with the distal airways and surrounded by fibrosis (Figure 1). In each biopsy, three distal airways, and in the case of UIP biopsies, three honeycomb cysts, were evaluated. In each area, quantification of the proportion of MUC5Band MUC5AC-positive cells, respectively, was evaluated in six randomly selected fields, each containing 100 airway (or honeycomb cyst, as appropriate) epithelial cells, by an observer blinded to clinical details (C.C.). Positive cells were defined as brown-stained elements, a sign of the antibody reaction with MUC5B or MUC5AC. Preliminary experiments showed that this sample size minimized the coefficient of variation and that the manual readings did correlate well with image analysis (ImageJ Threshold Color plugin), with absolute intraclass correlation coefficients of 0.81 (95% confidence interval, 0.71–0.87) for MUC5AC and 0.72 (95% confidence interval, 0.57–0.81) for MUC5B. To compare manual counts of MUC5B and MUC5AC staining across patterns, multilevel mixed-effects linear regression was performed, using a model in which patients were analyzed as random effect variables, with airways (or honeycomb cysts) and fields nested into patients (Stata 12, College Station, TX). In IPF/UIP distal airways, the proportion of MUC5B cells was more than twofold higher compared with control, i-NSIP, and SSc-NSIP distal airways (P, 0.0001 for all comparisons), even after adjustment for age, sex, and smoking status on multivariate analysis, whereas the proportion of MUC5B cells in honeycomb cysts was similar to control airways (Table 1 and Figure 1). No significant differences were observed in MUC5B expression between distal airways of SSc-NSIP, i-NSIP, and controls. In contrast, the proportion of MUC5AC epithelial cells in IPF/UIP distal airways was similar to control biopsies, whereas both i-NSIP and SSc-NSIP distal airways were characterized by significantly lower percentages of MUC5ACpositive cells (P, 0.001 vs. controls and UIP), even after adjustment for age, sex, and smoking history. In IPF/UIP honeycomb cysts, the proportion of MUC5AC-positive cells was significantly lower than in distal airways of control biopsies (P = 0.004). The higher expression of MUC5B in IPF/UIP distal airways when compared with control lungs is in keeping with the findings of Seibold and colleagues (12). However, we did not observe increased expression of MUC5AC in IPF/UIP distal airways compared with controls as described by Seibold and colleagues, a discrepancy that could at least partially be related to differing staining techniques. The relative sensitivity of immunofluorescence, used by Seibold and colleagues, and immunoperoxidase, used in this study, in analyzing mucin staining in formalin-fixed paraffin-embedded lung biopsies is not known, but it may be that sensitivity differs between the two techniques. In summary, we report that MUC5B overexpression appears to be specific to IPF/UIP, with twice the number of MUC5B cells seen in IPF/UIP distal airways compared with idiopathic and SScassociated fibrotic NSIP patterns. Further, the distal airways, rather than honeycomb cysts, appear to be the primary site of MUC5B overexpression in IPF lungs. Although we did not assess whether this was related to the MUC5B variant rs35705950, an association between the single-nucleotide polymorphism and overexpression of MUC5B in the distal airways, but not in honeycomb cysts of IPF lungs, was reported by Nakano and colleagues (16), This research project was supported by the European Respiratory Society, with a short-term fellowship grant awarded to C.C., and by the National Institute of Health Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust.

Effect of ambulatory oxygen on quality of life for patients with fibrotic lung disease (AmbOx): a prospective, open-label, mixed-method, crossover randomised controlled trial

BACKGROUND In fibrotic interstitial lung diseases, exertional breathlessness is strongly linked to health-related quality of life (HRQOL). Breathlessness is often associated with oxygen desaturation, but few data about the use of ambulatory oxygen in patients with fibrotic interstitial lung disease are available. We aimed to assess the effects of ambulatory oxygen on HRQOL in patients with interstitial lung disease with isolated exertional hypoxia. METHODS AmbOx was a prospective, open-label, mixed-method, crossover randomised controlled clinical trial done at three centres for interstitial lung disease in the UK. Eligible patients were aged 18 years or older, had fibrotic interstitial lung disease, were not hypoxic at rest but had a fall in transcutaneous arterial oxygen saturation to 88% or less on a screening visit 6-min walk test (6MWT), and had self-reported stable respiratory symptoms in the previous 2 weeks. Participants were randomly assigned (1:1) to either oxygen treatment or no oxygen treatment for 2 weeks, followed by crossover for another 2 weeks. Randomisation was by a computer-generated sequence of treatments randomly permuted in blocks of constant size (fixed size of ten). The primary outcome, which was assessed by intention to treat, was the change in total score on the Kings Brief Interstitial Lung Disease questionnaire (K-BILD) after 2 weeks on oxygen compared with 2 weeks of no treatment. General linear models with treatment sequence as a fixed effect were used for analysis. Patient views were explored through semi-structured topic-guided interviews in a subgroup of participants. This study was registered with ClinicalTrials.gov, number NCT02286063, and is closed to new participants with all follow-up completed. FINDINGS Between Sept 10, 2014, and Oct 5, 2016, 84 patients were randomly assigned, 41 randomised to ambulatory oxygen first and 43 to no oxygen. 76 participants completed the trial. Compared with no oxygen, ambulatory oxygen was associated with significant improvements in total K-BILD scores (mean 55·5 [SD 13·8] on oxygen vs 51·8 [13·6] on no oxygen, mean difference adjusted for order of treatment 3·7 [95% CI 1·8 to 5·6]; p<0·0001), and scores in breathlessness and activity (mean difference 8·6 [95% CI 4·7 to 12·5]; p<0·0001) and chest symptoms (7·6 [1·9 to 13·2]; p=0·009) subdomains. However, the effect on the psychological subdomain was not significant (2·4 [-0·6 to 5·5]; p=0·12). The most common adverse events were upper respiratory tract infections (three in the oxygen group and one in the no-treatment group). Five serious adverse events, including two deaths (one in each group) occurred, but none were considered to be related to treatment. INTERPRETATION Ambulatory oxygen seemed to be associated with improved HRQOL in patients with interstitial lung disease with isolated exertional hypoxia and could be an effective intervention in this patient group, who have few therapeutic options. However, further studies are needed to confirm this finding. FUNDING UK National Institute for Health Research.

Genetic predictors of systemic sclerosis-associated interstitial lung disease: a review of recent literature

The interplay between genetic and environmental factors is likely involved in the pathogenesis of systemic sclerosis (SSc). Interstitial lung disease associated in the context of SSc (SSc-ILD) is associated with significant morbidity, and is the leading cause of death in SSc. The spectrum of SSc-ILD severity is wide, ranging from patients with only limited and inherently stable pulmonary involvement, to those with extensive and progressive pulmonary fibrosis. In order to provide accurate prognostic information for patients, and to initiate appropriate monitoring and treatment regimens, the ability to identify patients at risk of developing severe ILD early in the disease course is crucial. Identification of genetic variants involved in disease pathogenesis can not only potentially provide diagnostic/prognostic markers, but can also highlight dysregulated molecular pathways for therapeutic targeting. A number of genetic associations have been established for susceptibility to SSc, but far fewer studies have investigated genetic susceptibility to SSc-ILD specifically. In this review we present a summary of the studies assessing genetic associations with SSc-ILD.

S45 MUC5B Genotype does not influence cough severity in IPF

Introduction Cough is a disabling symptom in IPF which causes significant reduction in quality of life. The mechanisms underlying cough in fibrotic lung disease are not well understood. Polymorphisms in the rs35105950 promoter region of the MUC5B gene have been shown to relate to risk of developing IPF and, in those with disease, have been shown to increase mucin expression in the small airways. A previous small study linked carriage of the minor T allele at rs35105950 with heightened cough in IPF. We sought to test this observation in a larger cohort of prospectively recruited individuals with IPF. Method MUC5B genotyping was performed on 117 prospectively recruited individuals with IPF. All subjects completed the Leicester Cough Questionnaire and full lung function at the time of their blood draw. Survival time from enrolment was censored at 1st May 2015. Statistical analyses were performed using SAS software. Results The mean age of subjects at enrolment was 68 ± 8.4 with 79% being male. Subjects had, on average, moderately severe disease with mean FVC 73.9 ± 19.7% predicted and DLco 41.7 ± 18.1% predicted. At baseline, the median (IQR) LCQ score was 16.0 (11.6–18.6) points. 43 subjects (36.8%) were homozygous at rs35105950 for the G allele, 62 (53.0%) were heterozygous and 12 (11.1%) were homozygous for the T allele. Despite a trend towards worse disease at baseline, homozygosity of the T allele was correlated with a significant survival advantage (422 days compared to GG, p = 0.0063). There was, however, no difference on univariate analysis in LCQ cough severity between groups based on MUC5B genotype (GG 14.6 ± 4.5, GT 15.3 ± 3.9 and TT 15.1 ± 3.5, p = 0.70). This finding did not change when correction was applied for baseline disease severity. Conclusions The severity of self-reported cough is worse in IPF than in other respiratory diseases. In contrast to one previous small study of 68 subjects, there is no relationship between patient reported cough and MUC5B genotype in our cohort. It therefore seems unlikely that the mechanism behind cough in fibrosing lung disease is related to mucin production. Further studies are required to determine the mechanisms underlying the heightened cough response observed in IPF.

P32 Role of non acid and proximal reflux in scleroderma-associated interstitial lung disease

Background Oesophageal involvement is extremely common in patients with scleroderma. This prospective observational study (NCT02136394) addresses the relationship between gastro-oesophageal reflux (GORD) and scleroderma-associated interstitial lung disease (SSc-ILD), and evaluates the clinical utility of noninvasive tests of microaspiration. Materials and methods We present preliminary results of the first 27 enrolled patients (median age 59 [min/max 35/79], median FVC = 74% [38/128%], median DLCO = 39% [21/72%], female 70%, diffuse SSc 33%). Collected clinical data included 24 hr impedance (carried out off PPI), respiratory (K-BILD and Leicester cough questionnaires) and GORD symptom questionnaires (UCLA SCTC GIT 2.0 Questionnaire, Reflux Disease Questionnaire RDQ), as well as full lung function test data. Pepsin levels were measured in saliva in all patients, and in a subset of 6 patients in bronchoalveolar lavage (BAL). Results Non acid reflux and proximal reflux were detected in 54% and 49% of patients, respectively. In the subgroup of patients with normal DeMeester score (i.e. global impedance index of acid exposure), 66% had non acid reflux episodes. The DeMeester score (median 14.2 [min/max 0.8/156]) was correlated with total scores GORD questionnaire scores (e.g. RDQ, r = 0.68 p = 0.003; GIT 2.0, r = 0.68 p = 0.004), but not with K-BILD, Leicester questionnaire, or saliva pepsin. Proximal reflux episodes were moderately correlated with the Leicester total score (r = -0.76 p = 0.002) and with saliva pepsin (r = 0.46 p = 0.05). Saliva pepsin (median concentration 2.34 ng/ml [2.34/12.4]) was correlated with the impedance cough index association (r = 0.53, p = 0.02). BAL pepsin was present in all six cases (median concentration 2.34 ng/ml [2.34/12.4]) and was correlated with FVC (r = -0.8, p = 0.04). Lung function test parameters were not correlated with saliva pepsin, but were significantly, if loosely, correlated with impedance measures of acid exposure in the recumbent position (e.g.% time of exposure, r = -0.43 p = 0.04). Conclusions Proximal and non acid reflux are highly prevalent in the SSc-ILD population and are associated with a high symptom burden. Pepsin is measurable in BAL of SSc-ILD patients and suggests microaspiration into the lungs, although larger numbers are needed to confirm these findings and define whether saliva pepsin measurement could represent a useful non invasive marker of microaspiration.