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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

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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.

Yes / Objectives: Diffuse idiopathic skeletal hyperostosis (DISH) has recurrently been associated with a rich diet (high in protein and higher trophic level foods); however, very few studies have investigated this link using carbon and nitrogen (δ13C and δ15N) stable isotope analysis. This paper explores the relationship between DISH and diet in two Roman urban communities by analyzing individuals with and without DISH. Materials and methods: δ13C and δ15N analysis carried out on collagen from 33 rib samples (No DISH: 27; early DISH: 4; DISH: 2) selected from individuals buried at the Romano-British site of Baldock (UK), 41 rib samples (No DISH: 38; early DISH: 3) from individuals from the Catalan Roman site of Santa Caterina (Barcelona, Spain). Additionally, six faunal samples from Baldock and seven from Santa Caterina were analyzed. Results: Standardized human isotope data from Santa Caterina show high δ15N probably associated to a diet combining terrestrial resources and freshwater fish. In contrast, isotope results from Baldock suggest a terrestrial-based diet. Individuals with DISH do not show isotopic ratios indicative of rich diet and there is no correlation between stage of DISH development and δ13C and δ15N. Conclusion: The results of this study suggest that individuals with DISH followed a similar or isotopically similar diet as those individuals without DISH in Baldock and in Santa Caterina and therefore, while DISH may have been influenced by individual's dietary habits, this is not reflected in their isotopic signature. / Institute of Life Sciences Research Studentship, University of Bradford, Bradford, UK

Adolescent idiopathic scoliosis (AIS) is a leading cause of pediatric structural spinal deformity, impacting up to 3-4% of adolescents globally. The lack of unifying mechanisms to explain its development and paucity of suitable animal or in-vitro study models have made it difficult to institute effective therapeutic approaches for AIS. Paraspinal muscles are crucial structures for mobility and stability of the spine, but their role in AIS is not clearly understood. Phenotypically, paraspinal muscle in AIS display myopathic features including fibrosis and fatty involution. However, the mechanism of development of paraspinal muscle phenotype and its contribution in AIS pathogenesis is not elucidated. This project aimed to understand the development of paraspinal muscle phenotype in patients with AIS via characterization of inflammatory phenotype, fibrosis and fatty involution, and autophagic machinery in paraspinal muscle. We demonstrated the presence of paraspinal muscle fibrosis and fatty involution on both concave and convex sides of the scoliotic curve. The potential crosstalk between TGF- family and mesenchymal progenitors expressing PDGFR- was identified and represent the crucial mechanism associated with development of muscle phenotype in AIS. We also demonstrated the upregulation of canonical TGF/Smad signaling pathways and Smad independent non-canonical TGFsignaling pathways including p38 and JNK (p46/54) MAPKs in paraspinal muscle on the concave versus convex side which may be associated with an enhanced fibrosis and fatty involution on the concave side in comparison to the convex side. Furthermore, the data suggest differential autophagy activation in paraspinal muscle in AIS with convex side demonstrating enhanced expression of autophagy markers in comparison to the concave side. The data also demonstrate that the Akt dependent inhibition of FoxO3A transcription factor could potentially lead to the suppression of expression of autophagy markers on the concave side in comparison to the convex side. The upregulation of fibrogenic and adipogenic pathways and suppression of expression of autophagy markers may associate with a more severe phenotype in paraspinal muscle on the concave side in comparison to the convex side. The assessment of muscle health in AIS opens therapeutic entry points to influence muscle phenotype in AIS which may impact the patient outcomes. / Thesis / Doctor of Philosophy (Medical Science)

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.