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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23783号 / 医博第4829号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 上野 英樹, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

Doctor of Philosophy / Department of Human Nutrition / Richard C. Baybutt / Weiqun George Wang / Although omega-3 fatty acids have been a hot issue in nutrition for years, there remains a paucity of research on the topic of omega-3 fatty acid and pulmonary fibrosis and the mechanism is still unclear. The purpose of this research is to investigate the preventive effects of flaxseed oil for bleomycin-induced pulmonary fibrosis in rats and to find the possible underlying mechanisms. There are two experiments demonstrated in this dissertation, one is with various doses of flaxseed oil in the diet (0, 2.5, 5, 7.5, 10, 12.5, and 15 % (w/w)), and the other is with different times of sacrificing animals after oropharyngeal bleomycin treatment (days 7 and 21). In the first study, three proteins including transforming growth factor-[beta] (TGF-[beta]), interleukin-1 (IL-1), and [alpha]-smooth muscle actin ([alpha]-SMA), commonly associated with fibrotic inflammation in the lung, were examined by Western blot and fatty acids composition of the diets and tissues were analyzed by gas chromatography (GC). Fifteen percent of flaxseed oil group significantly reduced septal and vascular thickness and fibrosis in the lung, and significant cardiac fibrosis in the heart. The amount of IL-1 and [alpha]-SMA decreased significantly as the amount of omega-3 fatty acids increased, whereas TGF-[beta] did not change significantly. The next study further reported the time-course effect and potential underlying mechanisms. Both interleukin-6 (IL-6), a protein associated with fibrotic inflammation in the lung, and renin, an enzyme related to renin-angiotensin system, were examined by Western blot. The time-dependent increase of IL-6 in response to bleomycin treatment was reversed by flaxseed oil diet. Although renin was not significantly different in the kidney, it suggested that the renin-angiotensin system may be involved locally. In addition, the profiles of fatty acids in both liver and kidney tissues as measured by lipidomics demonstrated a significant increase of omega-3: omega-6 ratio in the flaxseed oil-fed groups. Overall, these results indicated for the first time that the omega-3 fatty acids rich in flaxseed oil inhibited the formation of pulmonary fibrosis in a dose-dependent manner - however the moderate dose of flaxseed oil was most effective - via anti-inflammatory mechanisms, which appears associated with the modulated fatty acid composition in the tissues.

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.

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.

Idiopathic Pulmonary Fibrosis (IPF) is a deadly disease of unknown origin, which causes 80,000 deaths every year in the US and Europe combined. Unknown etiology and late diagnosis, combined with limited treatment options, contribute to a dismal survival rate of 3-5 years post diagnosis. Although molecular mechanisms underlying IPF pathogenesis and progression have been studied for over two decades, lack of in vivo models that recapitulate chronic, progressive, and irreversible nature of IPF have contributed to limited therapeutic success in clinical trials. Currently, only two drugs, Pirfenidone and Nintedanib, are approved for IPF treatment in the US, with their efficacy yet to be completely determined. Patients with IPF often observe lung infections, alveolar collapse, and respiratory failure, which are associated with focal edema and local hypoxia and contribute to development of hypoxemia associated with acute exacerbation of IPF (AE-IPF). In my thesis, I posit that hypoxic injury to the lung epithelium can initiate profibrotic signaling that can contribute to pathogenesis and progression of pulmonary fibrosis in vitro and in vivo. In my in silico studies, I analyzed human protein kinases to identify structural peculiarities that diversify their functions and highlight central hub kinases governing cell signaling. Using this approach, I identified Focal Adhesion Kinase 1 (FAK1) as a central hub kinase contributing to cytoskeletal remodeling. My proteomics and transcriptional studies defined in vitro effect of hypoxia in activation of lung epithelial cells. Using systems biology approaches, I identified interplay between transforming growth factor – β (TGF–β) signaling, hypoxia signaling, and FAK1 signaling. Further, my studies identified Galectin-1 as a novel mediator of hypoxia-induced pulmonary fibrosis. To mimic exacerbation of PF in patients, I developed a novel mouse model of exacerbated pulmonary fibrosis using subclinical bleomycin injury with chronic hypoxia. Further, to fill the existing requirement of an in vivo model of chronic PF, I characterized a triple transgenic mouse model that conditionally activates hypoxia signaling in the lung epithelial cells and causes progressive PF over a span of 12 weeks. Lastly, I performed RNA-Seq experiments on primary AEC2s isolated from our transgenic mouse model to identify a hypoxia-mediated profibrotic role of microRNA-96 in down-regulation of PTEN, a tumor suppressor and anti-fibrotic protein. In conclusion, my studies established in vitro and in vivo roles of hypoxia in profibrotic activation of lung epithelium and identifies FAK1 and Gal-1 as key drivers of hypoxia-mediated fibrosis, which should be further evaluated in animal and human studies to determine their therapeutic potential.

Chronic Obstructive Pulmonary Disease (COPD) is the 3rd leading cause of death in the US, and idiopathic pulmonary fibrosis (IPF), a type of Interstitial Lung Disease (ILD), is a fast acting, irreversible disease that leads to mortality within 3-5 years. RNA-sequencing provides the opportunity to quantitatively examine the sequences of millions mRNAs, and offers the potential to gain unprecedented insights into the structure of chronic non-malignant lung disease transcriptome. By identifying changes in splicing and novel loci expression associated with disease, we may be able to gain a better understanding of their pathogenesis, identify novel disease-specific biomarkers, and find better targets for therapy. Using RNA-seq data that our group generated on 281 human lung tissue samples (47=Control, 131=COPD, 103=ILD), I initially defined the transcriptomic landscape of lung tissue by identifying which genes were expressed in each tissue sample. I used a mixture model to separate genes into reliable and not reliable expression. Next, I employed reads that overlapped splice junctions in a linear model interaction term to identify disease-specific differential splicing. I identified alternatively spliced genes between control and disease tissues and validated three (PDGFA, NUMB, SCEL) of these genes with qPCR and nanostring (a hybridization-based barcoding technique used to quantify transcripts). Finally, I implemented and improved a pipeline to perform transcriptome assembly using Cufflinks that led to the identification of 1,855 novel loci that did not overlap with UCSC, Vega, and Ensembl annotations. The loci were classified into potential coding and non-coding loci (191 and 1,664, respectively). Expression analysis revealed that there were 120 IPF-associated and 10 emphysema-associated differentially expressed (q < 0.01) novel loci. RNA-seq provides a high-resolution transcript-level view of the pulmonary transcriptome and its modification in lung disease. It has enabled a new understanding of the lung transcriptome structure because it measures not only the transcripts we know but also the ones we do not know. The approaches and improvements I have employed have identified these novel targets and make possible further downstream functional analysis that could identify better targets for therapy and lead to an even better understanding of chronic lung disease pathogenesis. / 2031-01-01T00:00:00Z