An estimated 45% of all deaths can be attributed to various chronic fibroproliferative diseases. Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung disease which is characterized by progressive decline in lung function. While the pathogenesis of IPF is not fully understood, alternatively activated macrophages (M2) have been implicated as a key contributor to the fibrotic process. The plasticity of macrophages in vivo challenges the ability to specifically target the M2 macrophage phenotype across species. Previous bioinformatic analysis from our lab identified Dectin-1/Clec7a as a unique marker of M2 macrophages in both human and murine model systems. The expression of the transmembrane receptor Dectin-1 has not been elucidated in the context of pulmonary fibrosis. To prevent the progression of fibrosis by targeting alternatively activated macrophages, we investigated the expression of Dectin-1 in IPF and an experimental model of fibrotic lung disease. Our data demonstrated that while protein expression of Dectin-1 was increased in archived lung tissues of patients with IPF, mRNA expression of this receptor was downregulated in the tissues of these IPF patients. Gene expression of Dectin-1 was shown to be increased in monocyte-derived macrophages, further suggesting a circulatory component contributing to lung fibrosis. As expected, we confirmed that Dectin-1 was highly expressed past the injury phase of the bleomycin-model of induced pulmonary fibrosis which aligns with the increased immune infiltrates at this time point. Preliminary work into the time dependency of the resolution phase of the bleomycin-induced model of lung fibrosis was shown. All in all, our data suggests that Dectin-1 may be a useful marker in characterizing and differentiating phenotypes of macrophages implicated in the fibrotic process. Future efforts aim to gain insight into the functional requirement of Dectin-1 in the alternative activation of profibrotic macrophages to identify novel therapeutic targets for fibrotic lung disease. / Thesis / Master of Science (MSc)

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown pathogenesis characterized by scarring of the lung and declining respiratory function. Originating from bone marrow, circulating monocytes can be recruited into the lung tissue and polarized toward the alternatively activated (M2) profibrotic macrophage phenotype. Recent literature has shown that cluster of differentiation 14 positive (CD14+) monocytes are more abundant in IPF patient blood and are associated with disease outcome and acute exacerbation. Additionally, a 52-gene risk profile from peripheral blood mononuclear cells for outcome prediction in IPF was recently published. Here, we began by characterizing macrophages in human IPF lung tissue. We then assembled a biobank and examined transcriptomic characteristics of blood-derived circulating monocytes from IPF patients. Various histological assessments were completed on a tissue microarray including lung biopsies from 24 IPF patients and 17 controls, to characterize M2 macrophage expression in human tissue. Whole blood samples were collected from 50 IPF patients and 12 control subjects. CD14+ monocytes were isolated and mRNA was extracted for bulk RNA sequencing. Data were analyzed for differential expression (DE), and Gene Set Enrichment Analysis (GSEA) was performed to examine enrichment of the previously published 52-gene risk profile in our dataset. We found that M2 macrophage expression was increased in IPF lung tissue compared to controls. CD14+ monocyte levels were significantly elevated in IPF patients in our cohort compared to control participants, and was negatively correlated with forced vital capacity (FVC). DE analysis comparing IPF and control monocytes yielded a 35-gene signature, with 16 up-regulated genes and 19 down-regulated genes. When comparing the signature related to long transplant-free survival from the published dataset to our data, GSEA demonstrated that this signature is enriched in donors from our dataset, supporting concurrence between the meanings of the two datasets. Overall, these results provide insight to identify targets to modulate monocyte/macrophage function in IPF and potentially affect progressive disease. / Thesis / Master of Science (MSc) / Idiopathic pulmonary fibrosis (IPF) is a disease of unknown cause that results in excessive scarring of the lungs and progressive impairment in lung function. We believe that white blood cells called monocytes and macrophages play a key role in the development and progression of this disease. Overall, it is thought that monocytes, which circulate in the blood, enter the lung tissue and become macrophages. These macrophages then lead to the formation of scar tissue, which is characteristic to IPF. In order to better understand how these cells contribute to IPF, we studied their properties in blood and lung biopsies from IPF patients. We found significant differences between monocytes/macrophages in IPF than those in healthy controls, that may help explain disease progression. We hope that these findings will provide insight into causes of the IPF, and potential avenues for therapeutic intervention.

BACKGROUND: The course of disease for patients with idiopathic pulmonary fibrosis (IPF) is highly heterogeneous. Prognostic models rely on demographic and clinical characteristics and are not reproducible. Integrating data from genomic analyses may identify novel prognostic models and provide mechanistic insights into IPF. METHODS: Total RNA of peripheral blood mononuclear cells was subjected to microarray profiling in a training (45 IPF individuals) and two independent validation cohorts (21 IPF/10 controls, and 75 IPF individuals, respectively). To identify a gene set predictive of IPF prognosis, we incorporated genomic, clinical, and outcome data from the training cohort. Predictor genes were selected if all the following criteria were met: 1) Present in a gene co-expression module from Weighted Gene Co-expression Network Analysis (WGCNA) that correlated with pulmonary function (p < 0.05); 2) Differentially expressed between observed "good" vs. "poor" prognosis with fold change (FC) >1.5 and false discovery rate (FDR) < 2 %; and 3) Predictive of mortality (p < 0.05) in univariate Cox regression analysis. "Survival risk group prediction" was adopted to construct a functional genomic model that used the IPF prognostic predictor gene set to derive a prognostic index (PI) for each patient into either high or low risk for survival outcomes. Prediction accuracy was assessed with a repeated 10-fold cross-validation algorithm and independently assessed in two validation cohorts through multivariate Cox regression survival analysis. RESULTS: A set of 118 IPF prognostic predictor genes was used to derive the functional genomic model and PI. In the training cohort, high-risk IPF patients predicted by PI had significantly shorter survival compared to those labeled as low-risk patients (log rank p < 0.001). The prediction accuracy was further validated in two independent cohorts (log rank p < 0.001 and 0.002). Functional pathway analysis revealed that the canonical pathways enriched with the IPF prognostic predictor gene set were involved in T-cell biology, including iCOS, T-cell receptor, and CD28 signaling. CONCLUSIONS: Using supervised and unsupervised analyses, we identified a set of IPF prognostic predictor genes and derived a functional genomic model that predicted high and low-risk IPF patients with high accuracy. This genomic model may complement current prognostic tools to deliver more personalized care for IPF patients.

INTRODUÇÃO: As doenças pulmonares intersticiais fibrosantes (DPIFs) se caracterizam por dispneia, intolerância aos esforços e prejuízo da qualidade de vida. Apesar de existirem vários mecanismos implicados, a fisiopatologia da dispneia e limitação aos esforços não é completamente elucidada. A disfunção da musculatura ventilatória tem sido postulada como um fator envolvido. O objetivo do estudo foi investigar a ocorrência de disfunção muscular ventilatória em pacientes com DPIF comparados a indivíduos sadios, e correlacionar a disfunção muscular com a qualidade de vida, dispneia e intolerância ao exercício. METODOLOGIA: Foi realizado um estudo prospectivo, caso-controle envolvendo 62 indivíduos, sendo 31 pacientes com DPIF e 31 voluntários sadios. Os indivíduos foram avaliados em 2 visitas. Na visita 1 foram avaliados o grau de dispneia (escala de MRCm), a qualidade de vida (SGRQ), a função pulmonar, e o desempenho num teste de caminhada de 6 minutos (TC6M) além de caracterização da mobilidade e espessura do diafragma ao ultrassom (US). Na visita 2, foram avaliadas:1) a força muscular ventilatória estática volitiva (PImáx, PEmáx, SNIP, PesSniff, PgaSniff, PdiSniff, e PgaTosse) e não volitiva através da estimulação magnética bilateral do nervo frênico (TwPes, TwPga e TwPdi) e das raízes dorsais em T10 (TwT10Pga); 2) a sincronia toracoabdominal (por pletismografia de indutância); 3) o recrutamento dos músculo inspiratórios (eletromiografia de superfície do musculo escaleno) e expiratórios (eletromiografia de superfície do musculo obliquo externo). A seguir foi realizado um teste de exercicio cardiopulmonar (TECP) em cicloergômetro limitado por sintomas. As medidas de força muscular não volitiva foram repetidas após o TECP para investigar a ocorrência de fadiga muscular ventilatória. RESULTADOS: os pacientes com DPIFs apresentavam: dispneia aos esforços; limitação do desempenho no TC6M e prejuízo de qualidade de vida. Os pacientes com DPIF apresentaram redução da mobilidade diafragmática na respiração profunda, aumento da espessura na CRF e redução da fração de espessamento do diafragma ao US. Não houve diferenças entre pacientes e controles na força muscular volitiva e não volitiva e na proporção de fadiga ins e expiratória após o esforço. Contudo, os pacientes apresentaram fadiga ventilatória em cargas menores de exercicio. Nos pacientes com DPIF houve uma redução no desempenho do exercicio associada a uma limitação ventilatória, dessaturação e dispneia. Os pacientes com DPIF apresentaram uma proporção maior de assincronia no pico do exercício além de maior recrutamento do musculo escaleno. As relações entre a força ventilatória inspiratória e o os volumes pulmonares indicaram um desacoplamento neuromecânico (DNM) que se correlacionou com a dispneia nos pacientes com DPIF. CONCLUSÕES: Os pacientes com DPIF apresentam disfunção muscular ventilatória ao repouso caracterizado pela redução da mobilidade do diafragma na respiração profunda, aumento da espessura e redução da fração de espessamento. Ao esforço, na DPIF, a disfunção muscular ventilatória foi caracterizada pela ocorrência de fadiga ventilatória em baixas cargas de exercicio, recrutamento predominante dos músculos inspiratórios acessórios, assincronia toracoabdominal e desacoplamento neuromecânico que contribuíram para limitação do desempenho e dispneia / INTRODUCTION: fibrosing interstitial lung diseases (FILDs) are characterized by dyspnea, exercise intolerance and impaired quality of life. While there are several mechanisms involved, the occurrence of dyspnea and exercise limitation is not fully elucidated. The dysfunction of the respiratory muscles has been postulated as a contributing factor. The aim of the study was to investigate the occurrence of respiratory muscle dysfunction in patients with FILDs compared to healthy subjects and to correlate respiratory muscle dysfunction with quality of life, dyspnea and exercise intolerance. METHODS: A prospective, case-control study involving 62 subjects, 31 patients with FILD and 31 healthy volunteers. Subjects were evaluated in two visits. At visit 1, subjects underwent clinical evaluation to access dyspnea (MRCm), quality of life (SGRQ), pulmonary function and also characterization of mobility and thickness of the diaphragm on ultrasound (US). Subjects performed a 6-minute walk test (6MWT). In the second visit were evaluated: 1) maximum static respiratory pressures through volitional (MIP, MEP, SNIP, PesSniff, PgaSniff, PdiSniff and PgaCough) and non-volitional methods - cervical Twitchs (TwPes, TwPga and TwPdi) and T10 Twitchs (TwT10Pga); 2) thoracoabdominal synchrony (by respiratory inductance plethysmography); 3) recruitment of inspiratory muscle (surface electromyography of scalene muscle) and expiratory (surface electromyography of the external oblique muscle). Then, subjects performed an incremental cardiopulmonary exercise testing (CPET). The non-volitional muscle strength measures were repeated after the CPET to investigate the occurrence of fatigue. RESULTS: The patients with FILDs exhibited dyspnea on exertion; limited performance on 6MWT and impaired quality of life. On ultrasound, patients with FILD had decreased diaphragmatic mobility during deep breathing, increased thickness in the functional residual capacity (FRC) and reduced diaphragm thickness fraction. Between patients and controls, there were no differences in volitional and non-volitional strength and in the occurrence of respiratory fatigue. However, patients presented respiratory fatigue under lower exercise loads. In patients with FILD there was a decrease in exercise performance associated with ventilatory limitation, desaturation and dyspnea. Patients with FILD had a higher proportion of asynchrony at exercise peak and greater recruitment of the scalene muscle. In patients with FILD, higher inspiratory effort- displacement ratios indicated a neuromechanical uncoupling (DNM) that correlated with dyspnea. CONCLUSIONS: Patients with FILD exhibited respiratory muscle dysfunction at rest characterized by the reduction of diaphragmatic mobility in deep breathing, increased thickness on FRC and reduced thickness fraction. In FILD, exercise was associated with respiratory muscle dysfunction characterized by the occurrence of respiratory fatigue, thoracoabdominal asynchrony, greater recruitment of inspiratory muscles and neuromechanical uncoupling that contributed to limiting the performance and dyspnea

INTRODUÇÃO: As doenças pulmonares intersticiais fibrosantes (DPIFs) se caracterizam por dispneia, intolerância aos esforços e prejuízo da qualidade de vida. Apesar de existirem vários mecanismos implicados, a fisiopatologia da dispneia e limitação aos esforços não é completamente elucidada. A disfunção da musculatura ventilatória tem sido postulada como um fator envolvido. O objetivo do estudo foi investigar a ocorrência de disfunção muscular ventilatória em pacientes com DPIF comparados a indivíduos sadios, e correlacionar a disfunção muscular com a qualidade de vida, dispneia e intolerância ao exercício. METODOLOGIA: Foi realizado um estudo prospectivo, caso-controle envolvendo 62 indivíduos, sendo 31 pacientes com DPIF e 31 voluntários sadios. Os indivíduos foram avaliados em 2 visitas. Na visita 1 foram avaliados o grau de dispneia (escala de MRCm), a qualidade de vida (SGRQ), a função pulmonar, e o desempenho num teste de caminhada de 6 minutos (TC6M) além de caracterização da mobilidade e espessura do diafragma ao ultrassom (US). Na visita 2, foram avaliadas:1) a força muscular ventilatória estática volitiva (PImáx, PEmáx, SNIP, PesSniff, PgaSniff, PdiSniff, e PgaTosse) e não volitiva através da estimulação magnética bilateral do nervo frênico (TwPes, TwPga e TwPdi) e das raízes dorsais em T10 (TwT10Pga); 2) a sincronia toracoabdominal (por pletismografia de indutância); 3) o recrutamento dos músculo inspiratórios (eletromiografia de superfície do musculo escaleno) e expiratórios (eletromiografia de superfície do musculo obliquo externo). A seguir foi realizado um teste de exercicio cardiopulmonar (TECP) em cicloergômetro limitado por sintomas. As medidas de força muscular não volitiva foram repetidas após o TECP para investigar a ocorrência de fadiga muscular ventilatória. RESULTADOS: os pacientes com DPIFs apresentavam: dispneia aos esforços; limitação do desempenho no TC6M e prejuízo de qualidade de vida. Os pacientes com DPIF apresentaram redução da mobilidade diafragmática na respiração profunda, aumento da espessura na CRF e redução da fração de espessamento do diafragma ao US. Não houve diferenças entre pacientes e controles na força muscular volitiva e não volitiva e na proporção de fadiga ins e expiratória após o esforço. Contudo, os pacientes apresentaram fadiga ventilatória em cargas menores de exercicio. Nos pacientes com DPIF houve uma redução no desempenho do exercicio associada a uma limitação ventilatória, dessaturação e dispneia. Os pacientes com DPIF apresentaram uma proporção maior de assincronia no pico do exercício além de maior recrutamento do musculo escaleno. As relações entre a força ventilatória inspiratória e o os volumes pulmonares indicaram um desacoplamento neuromecânico (DNM) que se correlacionou com a dispneia nos pacientes com DPIF. CONCLUSÕES: Os pacientes com DPIF apresentam disfunção muscular ventilatória ao repouso caracterizado pela redução da mobilidade do diafragma na respiração profunda, aumento da espessura e redução da fração de espessamento. Ao esforço, na DPIF, a disfunção muscular ventilatória foi caracterizada pela ocorrência de fadiga ventilatória em baixas cargas de exercicio, recrutamento predominante dos músculos inspiratórios acessórios, assincronia toracoabdominal e desacoplamento neuromecânico que contribuíram para limitação do desempenho e dispneia / INTRODUCTION: fibrosing interstitial lung diseases (FILDs) are characterized by dyspnea, exercise intolerance and impaired quality of life. While there are several mechanisms involved, the occurrence of dyspnea and exercise limitation is not fully elucidated. The dysfunction of the respiratory muscles has been postulated as a contributing factor. The aim of the study was to investigate the occurrence of respiratory muscle dysfunction in patients with FILDs compared to healthy subjects and to correlate respiratory muscle dysfunction with quality of life, dyspnea and exercise intolerance. METHODS: A prospective, case-control study involving 62 subjects, 31 patients with FILD and 31 healthy volunteers. Subjects were evaluated in two visits. At visit 1, subjects underwent clinical evaluation to access dyspnea (MRCm), quality of life (SGRQ), pulmonary function and also characterization of mobility and thickness of the diaphragm on ultrasound (US). Subjects performed a 6-minute walk test (6MWT). In the second visit were evaluated: 1) maximum static respiratory pressures through volitional (MIP, MEP, SNIP, PesSniff, PgaSniff, PdiSniff and PgaCough) and non-volitional methods - cervical Twitchs (TwPes, TwPga and TwPdi) and T10 Twitchs (TwT10Pga); 2) thoracoabdominal synchrony (by respiratory inductance plethysmography); 3) recruitment of inspiratory muscle (surface electromyography of scalene muscle) and expiratory (surface electromyography of the external oblique muscle). Then, subjects performed an incremental cardiopulmonary exercise testing (CPET). The non-volitional muscle strength measures were repeated after the CPET to investigate the occurrence of fatigue. RESULTS: The patients with FILDs exhibited dyspnea on exertion; limited performance on 6MWT and impaired quality of life. On ultrasound, patients with FILD had decreased diaphragmatic mobility during deep breathing, increased thickness in the functional residual capacity (FRC) and reduced diaphragm thickness fraction. Between patients and controls, there were no differences in volitional and non-volitional strength and in the occurrence of respiratory fatigue. However, patients presented respiratory fatigue under lower exercise loads. In patients with FILD there was a decrease in exercise performance associated with ventilatory limitation, desaturation and dyspnea. Patients with FILD had a higher proportion of asynchrony at exercise peak and greater recruitment of the scalene muscle. In patients with FILD, higher inspiratory effort- displacement ratios indicated a neuromechanical uncoupling (DNM) that correlated with dyspnea. CONCLUSIONS: Patients with FILD exhibited respiratory muscle dysfunction at rest characterized by the reduction of diaphragmatic mobility in deep breathing, increased thickness on FRC and reduced thickness fraction. In FILD, exercise was associated with respiratory muscle dysfunction characterized by the occurrence of respiratory fatigue, thoracoabdominal asynchrony, greater recruitment of inspiratory muscles and neuromechanical uncoupling that contributed to limiting the performance and dyspnea

Background Idiopathic pulmonary fibrosis (IPF) is a devastating form of chronic lung injury of unknown aetiology characterised by progressive lung scarring. A diagnosis of definite IPF requires High Resolution Computed Tomography (HRCT) appearances indicative of usual interstitial pneumonia (UIP), or in patients with 'possible UIP' CT appearances, histological confirmation of UIP. However the proportion of such patients that undergo SLB varies, perhaps due to a perception of risk of biopsy and additive diagnostic value of biopsy in individual patients. We hypothesised that an underlying UIP pathological pattern may result in increased risk of death and aimed to explore this by comparing the risk of SLB in suspected idiopathic interstitial pneumonia, stratified according to HRCT appearance. Additionally we sought to determine the positive-predictive value of biopsy to diagnose IPF in patients with 'possible UIP HRCT' in our population. In patients with possible UIP who are not biopsied, the clinical value of bronchoalveolar lavage (BAL) is uncertain. We aimed to prospectively study the diagnostic and prognostic value of BAL differential cell count (DCC) in suspected IPF and determine the feasibility of repeat BAL and the relationship between DCC and disease progression in two successive BALs. We hypothesised that BAL DCC between definite and possible IPF was different and that baseline DCC and change in BAL DCC predicted disease progression. Alveolar macrophages (AMs) are an integral part of the lung's reparative mechanism following injury, however in IPF they contribute to pathogenesis by releasing pro-fibrotic mediators promoting fibroblast proliferation and collagen deposition. Expansion of novel subpopulations of pulmonary monocyte-like cells (PMLCs) has been reported in inflammatory lung disease. We hypothesised that a distinct AM polarisation phenotype would be associated with disease progression. We aimed to perform detailed phenotyping of AM and PMLCs in BAL in IPF patients. Several prognostic scoring systems and biomarkers have been described to predict disease progression in IPF but most were derived from clinical trial patients or tertiary referral centres and none have been validated in separate cohorts. We aimed to identify a predictive tool for disease progression utilising physiological, HRCT and serum biomarkers in a unique population of incident treatment naïve IPF patients. Methods Between 01/01/07 and 31/12/13, 611 consecutive incident patients with suspected idiopathic interstitial pneumonia (IIP) presented to the Edinburgh lung fibrosis clinic. Of these patients 222 underwent video-assisted thoracoscopic lung biopsy and histological pattern was determined according to ATS/ERS criteria. Post-operative mortality and complication rates were examined. Fewer than 2% received IPF-directed therapy and less than 1% of the cohort were lost to follow-up. Disease progression was defined as death or ≥10% decline in VC within 12 months of BAL. Cells were obtained by BAL and a panel of monoclonal antibodies; CD14, CD16, CD206, CD71, CD163, CD3, CD4, CD8 and HLA-DR were used to quantify and selectively characterise AMs, resident PMLCs, inducible PMLCs, neutrophils and CD4+/CD8+ T-cells using flow cytometry. Classical, intermediate and non-classical monocyte subsets were also quantified in peripheral blood. Potential biomarkers (n=16) were pre-selected from either previously published studies of IPF biomarkers or our hypothesis-driven profiling. Linear logistic regression was used on each predictor separately to assess its importance in terms of p-value of the associated weight, and the top two variables were used to learn a decision tree. Results Based on the 2011 ATS/ERS criteria, 87 patients were categorised as 'definite UIP', of whom 3 underwent SLB for clinical indications. IPF was confirmed in all 3 patients based on 2013 ATS/ERS/JRS/ALAT diagnostic criteria. 222 patients were diagnosed with 'possible UIP'; 55 underwent SLB, IPF was subsequently diagnosed in 37 patients, 4 were diagnosed with 'probable IPF' and 14 were considered 'not IPF'. In this group, 30 patients were aged 65 years or over and 25/30 (83%) had UIP on biopsy. 306 patients had HRCTs deemed 'inconsistent with UIP', SLB was performed in 168 patients. Post6 operative 30-day mortality was 2.2% overall, and 7.3% in the 'possible UIP' HRCT group. Patients with 'definite IPF' based on HRCT and SLB appearances had significantly better outcomes than patients with 'definite UIP' on HRCT alone (P=0.008, HR 0.44 (95% CI 0.240 to 0.812)). BAL DCC was not different between definite and possible UIP groups, but there were significant differences with the inconsistent with UIP group. In the 12 months following BAL, 33.3% (n=7/21) of patients in the definite UIP group and 29.5% (n=18/61) in the possible UIP group had progressed. There were no significant differences in BAL DCC between progressor and non-progressor groups. Mortality in patients with suspected IPF and a BAL DCC consistent with IPF was no different to those with a DCC inconsistent with IPF (P=0.425, HR 1.590 (95% CI 0.502 to 4.967)). There was no difference in disease progression in either group (P=0.885, HR 1.081 (95% CI 0.376 to 3.106)). There was no statistically significant difference in BAL DCC at 0 and 12 months in either group. There was no significant change in DCC between 0 and 12 month BALs between progressors and non-progressors. Repeat BAL was well tolerated in almost all patients. There was 1 death within 1 month of a first BAL and 1 death within 1 month of a second BAL; both were considered 'probably procedure-related'. AM CD163 and CD71 (transferrin receptor) expression were significantly different between groups (P < 0.0001), with significant increases in the IPF group vs non fibrotic ILD (P < 0.0001) and controls (P < 0.0001 and P < 0.001 respectively). CD71 expression was also significantly increased in the IPF progressor vs non-progressor group (P < 0.0001) and patients with high CD71 expression had significantly poorer survival than the CD71low group (P=0.040, median survival 40.5 and 75.6 months respectively). CD206 (mannose receptor) expression was also significantly higher in the IPF progressor vs non-progressor group (P=0.034). There were no differences in baseline BAL neutrophil, eosinophil or lymphocyte percentages between IPF progressor or non-progressor groups. The percentage of rPMLCs was significantly increased in BAL fluid cells of IPF patients compared to those with non-fibrotic ILD (P < 0.0001) and healthy controls (P < 0.05). Baseline rPMLC percentage was significantly higher in IPF progressors vs IPF non-progressors (P=0.011). Baseline BAL iPMLC:rPMLC ratio was also significantly different between IPF progressor and non-progressor groups (P=0.011). Disease progression was confidently predicted by a combination of clinical and serological variables. In our cohort we identified a predictive tool based on two key parameters, one a measure of lung function and one a single serum biomarker. Both parameters were entered into a decision tree, and when applied to our cohort yielded a sensitivity of 86.4%, specificity of 92.3%, positive predictive value of 90.5% and negative predictive value of 88.9%. We also applied previously reported predictive tools such as the GAP Index, du Bois score and CPI Index to the Edinburgh IPF cohort. Conclusions SLB can be of value in the diagnosis of ILD, however perhaps due to the perceived risks associated with the procedure, only a small percentage of patients undergo SLB despite recommendations that patients have histological confirmation of the diagnosis. Advanced age is a strong predictor for IPF, and in our cohort 83% of patients aged over 65 years with 'possible UIP' HRCT appearances, had UIP on biopsy. BAL and repeat BAL in IPF is feasible and safe (< 1.5% mortality). Of those that underwent repeat BAL, disease progression was not associated with a change in DCC. However, 22% of lavaged patients died or were deemed too frail to undergo a second procedure at 12 months. These data emphasise the importance of BAL in identifying a novel human AM polarisation phenotype in IPF. Our data suggests there is a distinct relationship between AM subtypes, cell-surface expression markers, PMLC subpopulations and disease progression in IPF. This may be utilised to investigate new targets for future therapeutic strategies. / Disease progression in IPF can be predicted by a combination of clinical variables and serum biomarker profiling. We have identified a unique prediction model, when applied to our locally referred, incident, treatment naïve cohort can confidently predict disease progression in IPF. IPF is a heterogeneous disease and there is a definite clinical need to identify 'personalised' prognostic biomarkers which may in turn lead to novel targets and the advent of personalised medicines.

O objetivo desse estudo foi avaliar o comprometimento de pequenas vias aéreas na Fibrose Pulmonar Idiopática (FPI) analisando curvas Pressão- Volume (PV) do sistema respiratório. Coletamos curvas PV de doze pacientes antes da biopsia pulmonar, que confirmou FPI em sete pacientes e Pneumonite de Hipersensibilidade em cinco. Todas as curvas foram ajustadas com modelo sigmóide, V = a + b / (1 + e -(P-c/d)), e exponencial V = A - B . e -k.P (aplicado apenas à parte superior). O modelo exponencial, apesar do bom ajuste à parte superior, não representou a parte inicial da curva, gerando parâmetros sem significado. O modelo sigmóide ajustou bem toda a curva e gerou parâmetros com significado fisiológico, que sugerem a presença de colapso de pequenas vias aéreas na FPI. / The objective of this study was to evaluate small airways compromise in Idiopathic Pulmonary Fibrosis (IPF) using pressure-volume (PV) curves of the respiratory system. We collected PV curves from twelve patients before lung biopsy, which confirmed IPF in seven patients and Hipersensitivity Pneumonia in five. All curves were fitted with a sigmoid model, V = a + b / (1 + e -(P-c/d)), and an exponential model, V = A - B . e -k.P (applied only to the superior part of the curve). The exponential model, despite having a good fit to the superior part of the curve, did not represent the initial part, and yielded parameters with no physiological meaning. The sigmoid model had a good fit to the entire curve and yielded parameters with physiological meaning, suggesting the presence of small airways collapse in IPF.

H2O2 generated by alveolar macrophages has been linked to the development pulmonary fibrosis, but little is known about its source, mechanism of production and exact role upon alveolar macrophage activation. In this study, we found that alveolar macrophages from asbestosis patients spontaneously produce high levels of H2O2 and have high expression of Cu,Zn-SOD. Cu,Zn-SOD localized to the mitochondrial intermembrane space (IMS) in asbestosis patients and asbestos induced translocation of Cu,Zn-SOD to the IMS. This process was unique to macrophages and dependent on functional mitochondrial respiration. The presence of at least one of the conserved cysteines was required for disulfide bond formation and mitochondrial translocation. These conserved cysteine residues were also necessary for enzyme activation and H2O2 generation. Cu,Zn-SOD-mediated H2O2 generation was inhibited by knockdown of the iron-sulfur protein, Rieske, in complex III. The role of Cu,Zn-SOD was biologically relevant as Cu,Zn-SOD-/- mice generated significantly less H2O2, had less oxidative stress, and were protected from developing pulmonary fibrosis. This protective mechanism is closely related to the alveolar macrophage activation and polarization in Cu,Zn-SOD-/- mice, as they had a dominant pro-inflammatory phenotype. Macrophages not only initiate and accentuate inflammation after tissue injury, but they are also involved in resolution and repair. The pro-inflammatory M1 macrophages have microbicidal and tumoricidal activity, whereas the M2 macrophages are involved in tumor progression and tissue remodeling, and can be pro-fibrotic in certain settings. We demonstrate that overexpression of Cu,Zn-SOD promoted macrophages polarization into an M2 phenotype. Furthermore, overexpression of Cu,Zn-SOD in mice resulted in a pro-fibrotic environment and accelerated the development of pulmonary fibrosis. The mechanism which Cu,Zn-SOD-mediated H2O2 utilizes to modulate macrophage M2 polarization is through redox regulation of a critical cysteine in STAT6. The polarization process, at least partially, was regulated by epigenetic modulation. We show that STAT6 was indispensable for Cu,Zn-SOD-mediated M2 polarization. STAT6 upregulated Jmjd3, a histone H3 lysine 27 demethylase, and initiated M2 gene transcriptional activation. Targeting STAT6 with leflunomide, which can reduce cellular ROS production and inhibit STAT6 phosphorylation, abolished M2 polarization and ameliorated the fibrotic development. Taken together, these observations provide a novel mechanism for the pathogenesis of pulmonary fibrosis whereby the antioxidant enzyme Cu,Zn-SOD plays a paradoxical role. The study highlights the importance of mitochondrial Cu,Zn-SOD and redox signals in macrophage polarization and fibrosis development. These observations demonstrate that the Cu,Zn-SOD-STAT6-Jmjd3 pathway is a novel regulatory mechanism for M2 polarization and that leflunomide is a potential therapeutic agent in the treatment of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease leading to decreased lung volume and eventual respiratory failure. At present, the median post-diagnosis lifespan is between three and six years. Myofibroblasts are collagen-secreting cells essential for wound healing, but also implicated in the fibroproliferation and extra cellular matrix deposition commonly seen in IPF. The nitric oxide (NO) signaling pathway is implicated in protomyofibroblast to myofibroblast transition and regulation. Previous work has shown that in pulmonary myofibroblasts, endothelial nitric oxide synthase (eNOS) is the primary NOS isoform expressed. The current study used cultured rat pulmonary myofibroblasts between passages two and five as a cell model. The cells were grown in normal growth media (DMEM + 10% FBS) or serum starved (DMEM + 0% FBS) to induce cellular differentiation. In this study, immunocytochemistry was used to show localization of eNOS is dependent on cellular differentiation, with protomyofibroblasts expressing eNOS primarily in the nucleus and protomyofibroblasts expressing eNOS in the perinuclear region. We also show catalytic activity and localization of eNOS are correlated by visualizing nitric oxide production in the cells using a permeable fluorescein chromophore. By using western blot analysis on fractionated cell lysates we found eNOS expressed in the nucleus under normal growth conditions. eNOS is at least partially regulated by intracellular calcium (Ca2+) and calmodulin (CaM). Western blot analysis using native eNOS and phospho-specific eNOS antibodies on fractionated cells treated with the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate (PMA) with and without addition of its antagonist ethylene glycol tetraacetic acid (EGTA) was conducted to investigate PKC’s role in eNOS regulation by phosphorylation. Indeed, PKC activation was found to mitigate expression in the nucleus, while inhibition of the activator restored the activity expression above basal levels. This finding correlates with previous data from our lab showing a decrease in activity in myofibroblasts treated with PMA and assayed amperometrically with an NO electrode.

La Fibrose Pulmonaire idiopathique (FPI) est une maladie fibroproliférative pour laquelle il n’existe aucun traitement efficace. Les mécanismes à l’origine de cette pathologie sont méconnus et impliquent plusieurs types cellulaires et facteurs de croissance, comme le TGF-β responsable de la différenciation de fibroblastes en myofibroblastes. Pour mieux comprendre ces mécanismes physiopathologiques, nous nous sommes intéressés à l’implication des miARN dans ce processus. Une analyse par puces à ADN de l’ensemble des miARN modulés dans des échantillons pulmonaires de souris, résistantes ou sensibles à la fibrose pulmonaire induite par la bléomycine, nous a permis d’identifier miR-199a-5p comme le meilleur candidat associé à la fibrose pulmonaire mais aussi fibrose rénale et hépatique. J’ai ensuite démontré que l’expression de miR-199a-5p était induite par le TGF-β in vitro, et que sa surexpression ectopique induisait la différenciation des fibroblastes. Une combinaison d’approche in silico et expérimentale, m’a permis d’identifier la Cavéoline-1 (CAV-1) comme cible de ce miARN. La CAV-1 est impliquée dans la dégradation du récepteur TGF-β. Ainsi, l’inhibition de CAV-1 par miR-199a-5p constitue une boucle de rétrocontrôle positif exacerbant la voie TGF-β. De manière intéressante, l’inhibition de miR-199a-5p in vitro régule la différenciation, la prolifération et la migration des fibroblastes pulmonaires par le TGF-β. Par ailleurs, nos résultats précliniques indiquent que l’inhibition de ce miARN diminue les marqueurs de fibrose, permettant d’envisager le développement de nouvelles approches thérapeutiques dans le traitement de la FPI et d’autres maladies fibroprolifératives. / Idiopathic Pulmonary Fibrosis (IPF) is a fibroproliferative disease with poor prognosis and for which no effective treatment exists. The mechanisms of this disease remain poorly understood and involve numerous cell types and growth factors such as TGF-β, which leads to the activation of lung fibroblasts into myofibroblasts; the key cell type driving the fibrogenic process. In this context, we focused the involvement of miRNAs in fibrosis process. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to lung fibrosis after bleomycin exposure. We identified miR- 199a-5p as the best candidate associated with lung fibrosis but also kidney and liver fibrosis. I observed that miR-199a-5p expression was induced upon TGF-β exposure, and that its ectopic expression was sufficient to promote the pathogenic activation of pulmonary fibroblasts. Using combination of targets miRNA prediction tools and a transcriptomic approach we identified the Caveolin-1 (CAV-1), a critical mediator of pulmonary fibrosis, as a specific target of miR-199a-5p. Thus, we shown that miR-199a-5p is a key effector of TGF-β signaling in lung fibroblasts by regulating CAV1. Interestingly, inhibition of miR-199a-5p in vitro prevents the differentiation, proliferation and migration of fibroblasts after TGF-β stimulation. Finally, our preclinical results indicate that inhibition of this miRNA decreases fibrosis markers. Thus, miR-199a-5p behaves as a major regulator of tissue fibrosis with therapeutic potency for the treatment of IPF and fibroproliferative diseases.