Gisela Lindahl

Pivotal role of connective tissue growth factor in lung fibrosis: MAPK-dependent transcriptional activation of type I collagen.

OBJECTIVE Connective tissue growth factor (CTGF; CCN2) is overexpressed in systemic sclerosis (SSc) and has been hypothesized to be a key mediator of the pulmonary fibrosis frequently observed in this disease. CTGF is induced by transforming growth factor beta (TGFbeta) and is a mediator of some profibrotic effects of TGFbeta in vitro. This study was undertaken to investigate the role of CTGF in enhanced expression of type I collagen in bleomycin-induced lung fibrosis, and to delineate the mechanisms of action underlying the effects of CTGF on Col1a2 (collagen gene type I alpha2) in this mouse model and in human pulmonary fibroblasts. METHODS Transgenic mice that were carrying luciferase and beta-galactosidase reporter genes driven by the Col1a2 enhancer/promoter and the CTGF promoter, respectively, were injected with bleomycin to induce lung fibrosis (or saline as control), and the extracted pulmonary fibroblasts were incubated with CTGF blocking agents. In vitro, transient transfection, promoter/reporter constructs, and electrophoretic mobility shift assays were used to determine the mechanisms of action of CTGF in pulmonary fibroblasts. RESULTS In the mouse lung tissue, CTGF expression and promoter activity peaked 1 week after bleomycin challenge, whereas type I collagen expression and Col1a2 promoter activity peaked 2 weeks postchallenge. Fibroblasts isolated from the mouse lungs 14 days after bleomycin treatment retained a profibrotic expression pattern, characterized by greatly elevated levels of type I collagen and CTGF protein and increased promoter activity. In vitro, inhibition of CTGF by specific small interfering RNA and neutralizing antibodies reduced the collagen protein expression and Col1a2 promoter activity. Moreover, in vivo, anti-CTGF antibodies applied after bleomycin challenge significantly reduced the Col1a2 promoter activity by approximately 25%. The enhanced Col1a2 promoter activity in fibroblasts from bleomycin-treated lungs was partly dependent on Smad signaling, whereas CTGF acted on the Col1a2 promoter by a mechanism that was independent of the Smad binding site, but was, instead, dependent on the ERK-1/2 and JNK MAPK pathways. The CTGF effect was mapped to the proximal promoter region surrounding the inverted CCAAT box, possibly involving CREB and c-Jun. In human lung fibroblasts, the human COL1A2 promoter responded in a similar manner, and the mechanisms of action also involved ERK-1/2 and JNK signaling. CONCLUSION Our results clearly define a direct profibrotic effect of CTGF and demonstrate its contribution to lung fibrosis through transcriptional activation of Col1a2. Blocking strategies revealed the signaling mechanisms involved. These findings show CTGF to be a rational target for therapy in fibrotic diseases such as SSc.

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.

Activation of key profibrotic mechanisms in transgenic fibroblasts expressing kinase-deficient type II transforming growth factor-beta receptor (T beta RII Delta k)

We have generated transgenic mice expressing a kinase-deficient type II transforming growth factor-β (TGFβ) receptor selectively on fibroblasts (TβRIIΔk-fib). These mice develop dermal and pulmonary fibrosis. In the present study we explore activation of TGFβ signaling pathways in this strain and examine the profibrotic properties of explanted transgenic fibroblasts including myofibroblast differentiation and abnormal metalloproteinase production. Gene expression profiles of littermate wild type or transgenic fibroblasts were compared using high-density gene arrays and validated by Taqman reverse transcriptase-PCR, Northern and Western blotting. Using a specific inhibitor (SD-208) we demonstrate that the abnormal phenotype of these cells is dependent upon TβRI kinase (ALK5) activity, and that transgenic fibroblasts show enhanced expression and activation of TGFβ together with increased levels of wild type TβRII. Moreover, we confirm that transgene expression is itself regulated by TGFβ and that expression at low levels facilitates signaling, whereas high level expression is inhibitory. For a subset of TGFβ responsive genes basal up-regulation is normalized or suppressed by exogenous recombinant TGFβ1 at time points coincident with increased transgene expression. These findings explain the profound refractoriness of TβRIIΔk-fib fibroblasts to exogenous TGFβ1, despite their activated phenotype. Thus, transgenic fibroblasts recapitulate many hallmark biochemical properties of fibrotic cells, including high level CTGF (CCN2) expression and type I collagen overproduction, altered MMP production, and myofibroblast differentiation. These cells also show an enhanced ability to contract collagen gel matrices. Our study demonstrates that altered high affinity TGFβ receptor function may lead to ligand-dependent activation of downstream signaling, and provides further evidence of a pivotal role for sustained TGFβ overactivity in fibrosis.

Fibroblast-specific perturbation of transforming growth factor β signaling provides insight into potential pathogenic mechanisms of scleroderma-associated lung fibrosis: Exaggerated response to alveolar epithelial injury in a novel mouse model

OBJECTIVE To explore increased susceptibility to fibrosis following experimental injury to alveolar epithelial cells (AECs) in a novel transgenic mouse model of scleroderma with fibroblast-specific perturbation of transforming growth factor beta (TGFbeta) signaling (TbetaRIIDeltak-fib mice). METHODS Wild-type (WT) and transgenic mice were injured with intratracheally administered saline or bleomycin, and the lungs were harvested for biochemical, histologic, and electron microscopic analysis. RESULTS Electron microscopy revealed AEC abnormalities in the lungs of untreated transgenic mice and bleomycin-treated WT mice; the lungs of transgenic mice treated with bleomycin showed severe epithelial damage. Compared with lungs from bleomycin-treated WT mice, lungs from bleomycin-treated transgenic mice demonstrated increased fibroproliferation, myofibroblast persistence, and impaired hyperplasia and increased apoptosis of type II AECs. The lungs from saline-treated transgenic mice and those from bleomycin-treated WT mice had phenotypic similarities, suggesting enhanced susceptibility to minor epithelial injury in the transgenic strain. The level of collagen was increased in the lungs from transgenic mice compared with that in the lungs from WT mice after treatment with either bleomycin or saline. Persistent fibrosis in bleomycin-treated transgenic mice was independent of ongoing neutrophil inflammation but was associated with impaired alveolar epithelial repair. CONCLUSION These results suggest that in the context of fibroblast-specific perturbation of TGFbeta signaling, even minor epithelial injury induces significant fibrosis. The model supports a central role for TGFbeta in determining fibrosis and demonstrates that lung fibroblasts may regulate the response of AECs to injury. Our findings provide insight into likely pathogenic mechanisms in scleroderma-associated pulmonary fibrosis.

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.

Cross-talk between MCP-3 and TGFβ promotes fibroblast collagen biosynthesis

Recent studies have demonstrated upregulation of monocyte chemoattractant protein-3 (MCP-3/CCL7) in fibrosis and have suggested that in addition to a major role in regulating leucocyte recruitment this chemokine may also promote extracellular matrix (ECM) overproduction by fibroblasts. In the present study we explore interplay between MCP-3 and transforming growth factor beta (TGFbeta), a potent profibrotic cytokine. We demonstrate that MCP-3 promotes activation of TGFbeta signalling pathways leading to increased type I collagen secretion. In addition we show that MCP-3 gene expression is stimulated by recombinant TGFbeta1, raising the possibility for synergy between these two mediators in the fibrotic microenvironment. Comparison of downstream signalling pathways that regulate collagen gene activation by both cytokines confirms the central role of MAPK pathway activation in mediating the effects of both factors. An additive effect of these two agonists was demonstrated by comparative microarray analysis for key TGFbeta regulated transcripts including PAI-1, OSF2 and IGFBP6. Together, our results confirm cross-talk between MCP-3 and TGFbeta that may be critical in the development of fibrosis.

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.

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.