Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule.

Progressive organ fibrosis accounts for one-third of all deaths worldwide, yet preclinical models that mimic the complex, progressive nature of the disease are lacking, and hence, there are no curative therapies. Progressive fibrosis across organs shares common cellular and molecular pathways involving chronic injury, inflammation, and aberrant repair resulting in deposition of extracellular matrix, organ remodeling, and ultimately organ failure. We describe the generation and characterization of an in vitro progressive fibrosis model that uses cell types derived from induced pluripotent stem cells. Our model produces endogenous activated transforming growth factor β (TGF-β) and contains activated fibroblastic aggregates that progressively increase in size and stiffness with activation of known fibrotic molecular and cellular changes. We used this model as a phenotypic drug discovery platform for modulators of fibrosis. We validated this platform by identifying a compound that promotes resolution of fibrosis in in vivo and ex vivo models of ocular and lung fibrosis

Unraveling disease mechanisms of different lung pathologies with single-cell RNA sequencing

The respiratory system is composed of different tissues with their respective cell types that together work in concert to perform air conductance and gas exchange. With the advent of single-cell RNA-sequencing (scRNA-seq), it is now possible to comprehensively interrogate the function of each individual cell in homeostatic and diseased states. In this dissertation, various roles of epithelial, mesenchymal, and immune cell types of the respiratory system in idiopathic pulmonary fibrosis (IPF) and corona virus disease 2019 (COVID-19) were investigated with scRNA-seq. IPF is a chronic interstitial lung disease characterized by the progressive scarring of the lung parenchyma. Previous studies that surveyed the cellular landscape of IPF lungs utilized explant lungs that reflect end-stage fibrosis. To uncover disease mechanisms of airway cell types in early-stage fibrosis, air-liquid interface (ALI) cultures of primary cells taken from newly diagnosed IPF patients were used. This identified proinflammatory epithelial cells, profibrotic basal cells, and primed fibroblasts as early-stage drivers of IPF. Treatment with antifibrotic compounds nintedanib, pirfenidone, and saracatinib fail to completely ameliorate the identified signatures. With the emergence of the COVID-19 pandemic and its extensive public health burden, it was imperative to understand the molecular mechanisms of viral entry and disease pathology to identify potential risk factors and therapeutic targets. In the early stages of the pandemic, viral entry factors ACE2, TMPRSS2, and FURIN were found to be expressed by a transient secretory cell type (differentiating from secretory to ciliated cell) of the airway mucosa and by alveolar type 2 cells of the alveolar epithelium. With further investigation of severe COVID-19, the early-stage of COVID-19 infection characterized itself with a hyperactivated immune response mediated by proinflammatory macrophages. On the other hand, late-stage COVID-19, especially those with acute respiratory distress syndrome (ARDS), was characterized by an accumulation of profibrotic macrophages and activated myofibroblasts that drove pulmonary scarring and fibrosis. Although IPF and COVID-19 are different diseases by their own right, they share a commonality in aberrant wound healing responses. Both diseases are characterized by tissue inflammation that is followed by a profibrotic phase. Unlike in IPF where the tissue remodeling is progressive and chronic, COVID-19 ARDS-associated fibrosis undergoes a resolution phase. Future studies comparing the cellular and transcriptional landscape of both conditions in early and late stages of disease will uncover pathogenic mechanisms and therapeutic targets of lung fibrosis. The application of high-resolution transcriptomic profiling techniques such as scRNA-seq permits the interrogation of individual cell types and their direct contribution to the development of diseases. Moreover, it allows the comparison and transfer of identified pathomechanisms across different pulmonary diseases and, in doing so, provides deeper and generalizable insights. As this field continues to evolve, it will undoubtedly continue to provide a deeper understanding of respiratory diseases.Das respiratorische System setzt sich aus verschiedenen Geweben und ihren zugrundeliegenden Zelltypen zusammen, die gemeinsam Luftaufnahme und Gasaustausch gewährleisten. Mit dem Aufkommen der Einzelzell-RNA-Sequenzierung (scRNA-seq) ist es nun möglich, die Funktion jeder einzelnen Zelle in homöostatischen und kranken Zuständen umfassend zu untersuchen. In dieser Dissertation wurden verschiedene Rollen von Epithel-, Mesenchymal- und Immunzelltypen des Atmungssystems bei idiopathischer Lungenfibrose (IPF) und der Coronavirus-Krankheit-2019 (COVID-19) mit scRNA-seq untersucht. IPF ist eine chronische interstitielle Lungenerkrankung, die durch eine fortschreitende Vernarbung des Lungenparenchyms gekennzeichnet ist. Frühere Studien, die die Zellkomposition von IPF-Lungen untersuchten, verwendeten Lungenexplantate, die das Endstadium der Fibrose widerspiegeln. Um Krankheitsmechanismen von Atemwegszelltypen im Frühstadium der Fibrose aufzudecken, wurden Air-Liquid-Interface (ALI)-Kulturen von primären Zellen verwendet, die frisch diagnostizierten IPF-Patienten entnommen wurden. Dabei wurden proinflammatorische Epithelzellen, profibrotische Basalzellen und aktivierte Fibroblasten als treibende Kräfte im Frühstadium der IPF identifiziert. Die Behandlung mit den antifibrotischen Wirkstoffen Nintedanib, Pirfenidon und Saracatinib führte nicht zu einer vollständigen Verbesserung der identifizierten Signaturen. Mit dem Beginn der COVID-19-Pandemie und ihrer großen Belastung für die öffentliche Gesundheit war es unerlässlich, die molekularen Mechanismen des Viruseintritts und der Krankheitspathologie zu verstehen, um potenzielle Risikofaktoren und therapeutische Ansätze zu identifizieren. In den frühen Stadien der Pandemie wurde festgestellt, dass die viralen Eintrittsfaktoren ACE2, TMPRSS2 und FURIN von einem vorübergehenden sekretorischen Zelltyp (der sich von sekretorischen zu ziliierten Zellen differenziert) der Atemwegsschleimhaut und von Typ-2 -Pneumozyten des Alveolarepithels exprimiert werden. Bei der weiteren Untersuchung von schweren COVID-19 Verläufen zeigte sich, dass das Frühstadium der COVID-19-Infektion durch eine hyperaktivierte Immunantwort charakterisiert ist, die durch proinflammatorische Makrophagen vermittelt wird. Andererseits war das Spätstadium der COVID-19-Infektion, insbesondere bei Patienten mit akutem Atemnotsyndrom (ARDS), durch eine Anhäufung von profibrotischen Makrophagen und aktivierten Myofibroblasten gekennzeichnet, die die pulmonale Narbenbildung und Fibrose vorantrieben. Obwohl es sich bei IPF und COVID-19 um unterschiedliche Krankheiten handelt, ähneln sie sich in ihrer gestörten Wundheilung. Beide Krankheiten sindS durch eine Gewebeentzündung gekennzeichnet, auf die eine profibrotische Phase folgt. Im Gegensatz zur IPF, bei der die Gewebeveränderung fortschreitend und chronisch ist, durchläuft die COVID-19 ARDS-assoziierte Fibrose eine Reparationsphase. Zukünftige Studien, die die zelluläre und transkriptionelle Landschaft beider Erkrankungen in frühen und späten Stadien vergleichen, werden pathogene Mechanismen und therapeutische Ansätze der Lungenfibrose aufdecken können. Die Anwendung hochauflösender transkriptomischer Sequenzierung wie scRNA-seq ermöglicht die Untersuchung einzelner Zelltypen und ihren Beitrag zur Entstehung von Krankheiten. Darüber hinaus ermöglicht sie den Vergleich und die Übertragbarkeit identifizierter Pathomechanismen über verschiedene Lungenkrankheiten hinweg und liefert so tiefere und generalisierbare Erkenntnisse. Da sich dieses Feld stetig weiter entwickelt, wird es zweifellos auch weiterhin zu einem tieferen Verständnis von Atemwegserkrankungen beitragen

A novel multi-network approach reveals tissue-specific cellular modulators of fibrosis in systemic sclerosis

BACKGROUND: Systemic sclerosis (SSc) is a multi-organ autoimmune disease characterized by skin fibrosis. Internal organ involvement is heterogeneous. It is unknown whether disease mechanisms are common across all involved affected tissues or if each manifestation has a distinct underlying pathology. METHODS: We used consensus clustering to compare gene expression profiles of biopsies from four SSc-affected tissues (skin, lung, esophagus, and peripheral blood) from patients with SSc, and the related conditions pulmonary fibrosis (PF) and pulmonary arterial hypertension, and derived a consensus disease-associate signature across all tissues. We used this signature to query tissue-specific functional genomic networks. We performed novel network analyses to contrast the skin and lung microenvironments and to assess the functional role of the inflammatory and fibrotic genes in each organ. Lastly, we tested the expression of macrophage activation state-associated gene sets for enrichment in skin and lung using a Wilcoxon rank sum test. RESULTS: We identified a common pathogenic gene expression signature-an immune-fibrotic axis-indicative of pro-fibrotic macrophages (MØs) in multiple tissues (skin, lung, esophagus, and peripheral blood mononuclear cells) affected by SSc. While the co-expression of these genes is common to all tissues, the functional consequences of this upregulation differ by organ. We used this disease-associated signature to query tissue-specific functional genomic networks to identify common and tissue-specific pathologies of SSc and related conditions. In contrast to skin, in the lung-specific functional network we identify a distinct lung-resident MØ signature associated with lipid stimulation and alternative activation. In keeping with our network results, we find distinct MØ alternative activation transcriptional programs in SSc-associated PF lung and in the skin of patients with an "inflammatory" SSc gene expression signature. CONCLUSIONS: Our results suggest that the innate immune system is central to SSc disease processes but that subtle distinctions exist between tissues. Our approach provides a framework for examining molecular signatures of disease in fibrosis and autoimmune diseases and for leveraging publicly available data to understand common and tissue-specific disease processes in complex human diseases

Mechanisms of myeloid cell recruitment and biomarker potential in interstitial lung diseases

Interstitial lung diseases (ILDs) are fibrotic disorders with chronic inflammation and fibrinogenesis leading to lung scaring and lung function decline. Ultimately, progressive pulmonary fibrosis results in altered pulmonary physiology, abnormal gas exchange, and organ failure. ILDs include known causes and idiopathic causes, as it is the case of idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP). The most detrimental type of ILD is IPF in which anti-fibrotic drugs (nintedanib and pirfenidone) only decrease disease progression. For other ILD types, corticoid treatment helps to decrease exacerbation. Currently, clinical trials are evaluating the applicability of anti-fibrotic drugs for treating non-IPF ILDs. Therefore, mechanistic insights and in-depth cell characterization during tissue injury and remodeling in ILD are of great interest in the respiratory medical field. Circulating immune cell populations have been suggested to play a critical role in ILDs. For instance, mononuclear phagocytes are involved in the initiation, repair and regeneration of pulmonary fibrosis. Moreover, the close interaction between circulating and lung tissue-resident immune cells is critical to contribute to tissue homeostasis or lead to disease. However, precise myeloid phenotypes (e.g. myeloid-derived suppressor cells and monocytes) and their mechanisms of recruitment in ILDs have not yet been explored. In the first results chapter of this thesis, myeloid-derived suppressor cells (MDSC) abundance and function were investigated for the first time in IPF patients. For that, peripheral blood of 170 patients including IPF, non-IPF ILD, chronic obstructive pulmonary diseases (COPD) and controls were collected to characterize and quantify MDSC by flow cytometry. Circulating MDSC in IPF and non-IPF ILD were increased when compared with control. Moreover, cross sectional and longitudinal analysis of the abundance of MDSC inversely correlated with pulmonary function test in IPF only. IPF patients with high number of MDSC showed downregulation of co-stimulatory T cells signals quantified by qRT-PCR. Furthermore, MDSC were able to suppress lymphocytes CD4+ and CD8+ cells proliferation in vitro. Last, CD33 CD11b double positive cells, suggestive of MDSC, were found in neighboring fibrotic niches of the IPF lungs. Taking together, these results show that MDSC are potential biomarker for IPF and are suppressing T cell responses. In the second results chapter, we aimed at analyzing monocyte phenotype and recruitment from the blood to the lung tissue in ILD. Importantly, CX3CR1 expression on immune cells has been demonstrated to increase fibrosis features. For that, flow cytometry analysis of circulating monocytes was performed in 105 subjects (83 ILD, and 22 controls). Monocyte localization and abundance in the lung was assessed by immunofluorescence and flow cytometry analysis. For receptor-ligand function and transmigration pattern, monocytes were isolated from blood and cultured either alone or with endothelial cells. Here, we showed that classical monocytes (CM) were increased, while non-classical monocytes (NCM) were decreased in ILD: NSIP, hypersensitivity pneumonitis (HP) and connective tissue disease associated with ILD (CTD-ILD) compared with controls. Monocytes abundance positively correlated with lung function. Fractalkine levels, the ligand of CX3CR1, were higher in lung tissue than in plasma in ILD and also co-localized with bronchial ciliated cells. Fractalkine enhanced endothelial transmigration of NCM in ILD only. Flow cytometry and immunofluorescence staining showed increased NCM in ILD. NCM-derived cells in the ILD lungs co-stained with CX3CR1, M2-like and phagocytic markers. In summary, we show that epithelial-derived fractalkine drives the migration of NCM-CX3CR1 which provides an interstitial scavenger and phagocytic myeloid cells population in fibrotic ILD lungs

The role of Slc7a11 in controlling extracellular and intracellular redox environments of lung fibroblasts - potential targets for intervention in aging and idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by extracellular matrix deposition by fibroblasts. Aging and oxidative stress increase the susceptibility to IPF. Redox couples, cysteine/cystine (Cys/CySS) and glutathione/glutathione disulfide (GSH/GSSG), and their redox potentials (Eh) quantify oxidative stress. Fibroblasts from old mice maintain more oxidized extracellular Eh(Cys/CySS) than young mice. Microarray shows down-regulation of Slc7a11 potentially mediates this age-related oxidation. Slc7a11 is the key component of system Xc-, an antiporter that imports CySS and exports glutamate. The first aim of this dissertation is to investigate the mechanistic link between Slc7a11 expression and extracellular Eh(Cys/CySS). The second aim is to evaluate the effects of aging on the redox states of intracellular proteins and whether Slc7a11 contributes to the age-dependent effects. The last aim is to compare SLC7A11 expression, extracellular Eh(Cys/CySS) and intracellular Eh(GSH/GSSG) between human lung fibroblasts from IPF and non-IPF donors and to explore their association with pro-fibrotic gene expression. Slc7a11 expression was manipulated by pharmacological and genetic methods. Reduced and oxidized forms of Cys residues were labelled by Iodoacetyl Tandem Mass Tags. The ratio of oxidized/reduced forms (i.e., redox state) of a Cys residue was determined by multiplexed tandem mass spectrometry. Eh(Cys/CySS) and Eh(GSH/GSSG) were more oxidized in conditioned media of old fibroblasts. Up-regulation of Slc7a11 reduced extracellular Eh(Cys/CySS) for old fibroblasts. Inhibition of GSH synthesis had no effect on the ability of cells to restore their extracellular Eh(Cys/CySS). Redox states of 151 proteins changed with aging. Slc7a11 over-expression restored redox states of 104 proteins. Ingenuity Pathway Analysis showed these 104 proteins were involved in pathways of protein translation initiation, ubiquitin-proteasome-mediated degradation and integrin-cytoskeleton-associated signaling. Slc7a11 expression was lower in IPF fibroblasts. Extracellular Eh(Cys/CySS) was more oxidized and expression of pro-fibrotic genes was higher in IPF fibroblasts. In conclusion, Slc7a11 is the key regulator of extracellular Eh(Cys/CySS). Its effects are independent of GSH synthesis. Aging results in changes of redox states of proteins involved in protein turnover and cytoskeleton dynamics. Up-regulating Slc7a11 restores changes of protein redox states due to aging. Decreased SLC7A11 might represent a susceptibility factor for developing tissue disrepair and fibrosis in IPF

ROLE OF NUDT21 MEDIATED ALTERNATIVE POLYADENYLATION AND HYALURONAN IN THE DEVELOPMENT OF PULMONARY HYPERTENSION

Pulmonary hypertension (PH) is a progressive disease with serious effects on quality of life and life expectancy of patients. PH is a complex disease that likely develops due to multiple influences, and no curative treatments exist for this disease. It has been shown that alternative polyadenylation (APA) due to depletion of Nudix Hydrolase 21(NUDT21) is involved in several disease states including the chronic lung disease idiopathic pulmonary fibrosis (IPF). Additionally, hyaluronan, an extracellular matrix glycosaminoglycan has been associated with PH. The role and mechanism of NUDT21 and hyaluronan have not yet been described in this disease. My results reveal that NUDT21 depletion and APA in pulmonary artery smooth muscle cells (PASMCs) is associated with phenotypic changes and PH. I I also show that hyaluronan and hyaluronan related genes play important roles in the development of Pulmonary Arterial Hypertension (PAH) and PH associated with IPF and combined pulmonary fibrosis and emphysema. I also identify 4-methylumbelliferone as an inhibitor of PH in this mechanism. These studies provide new mechanisms for understanding the development of PH and potential therapeutic targets

Mechanisms of myeloid cell recruitment and biomarker potential in interstitial lung diseases

Interstitial lung diseases (ILDs) are fibrotic disorders with chronic inflammation and fibrinogenesis leading to lung scaring and lung function decline. Ultimately, progressive pulmonary fibrosis results in altered pulmonary physiology, abnormal gas exchange, and organ failure. ILDs include known causes and idiopathic causes, as it is the case of idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP). The most detrimental type of ILD is IPF in which anti-fibrotic drugs (nintedanib and pirfenidone) only decrease disease progression. For other ILD types, corticoid treatment helps to decrease exacerbation. Currently, clinical trials are evaluating the applicability of anti-fibrotic drugs for treating non-IPF ILDs. Therefore, mechanistic insights and in-depth cell characterization during tissue injury and remodeling in ILD are of great interest in the respiratory medical field. Circulating immune cell populations have been suggested to play a critical role in ILDs. For instance, mononuclear phagocytes are involved in the initiation, repair and regeneration of pulmonary fibrosis. Moreover, the close interaction between circulating and lung tissue-resident immune cells is critical to contribute to tissue homeostasis or lead to disease. However, precise myeloid phenotypes (e.g. myeloid-derived suppressor cells and monocytes) and their mechanisms of recruitment in ILDs have not yet been explored. In the first results chapter of this thesis, myeloid-derived suppressor cells (MDSC) abundance and function were investigated for the first time in IPF patients. For that, peripheral blood of 170 patients including IPF, non-IPF ILD, chronic obstructive pulmonary diseases (COPD) and controls were collected to characterize and quantify MDSC by flow cytometry. Circulating MDSC in IPF and non-IPF ILD were increased when compared with control. Moreover, cross sectional and longitudinal analysis of the abundance of MDSC inversely correlated with pulmonary function test in IPF only. IPF patients with high number of MDSC showed downregulation of co-stimulatory T cells signals quantified by qRT-PCR. Furthermore, MDSC were able to suppress lymphocytes CD4+ and CD8+ cells proliferation in vitro. Last, CD33 CD11b double positive cells, suggestive of MDSC, were found in neighboring fibrotic niches of the IPF lungs. Taking together, these results show that MDSC are potential biomarker for IPF and are suppressing T cell responses. In the second results chapter, we aimed at analyzing monocyte phenotype and recruitment from the blood to the lung tissue in ILD. Importantly, CX3CR1 expression on immune cells has been demonstrated to increase fibrosis features. For that, flow cytometry analysis of circulating monocytes was performed in 105 subjects (83 ILD, and 22 controls). Monocyte localization and abundance in the lung was assessed by immunofluorescence and flow cytometry analysis. For receptor-ligand function and transmigration pattern, monocytes were isolated from blood and cultured either alone or with endothelial cells. Here, we showed that classical monocytes (CM) were increased, while non-classical monocytes (NCM) were decreased in ILD: NSIP, hypersensitivity pneumonitis (HP) and connective tissue disease associated with ILD (CTD-ILD) compared with controls. Monocytes abundance positively correlated with lung function. Fractalkine levels, the ligand of CX3CR1, were higher in lung tissue than in plasma in ILD and also co-localized with bronchial ciliated cells. Fractalkine enhanced endothelial transmigration of NCM in ILD only. Flow cytometry and immunofluorescence staining showed increased NCM in ILD. NCM-derived cells in the ILD lungs co-stained with CX3CR1, M2-like and phagocytic markers. In summary, we show that epithelial-derived fractalkine drives the migration of NCM-CX3CR1 which provides an interstitial scavenger and phagocytic myeloid cells population in fibrotic ILD lungs

Towards a global initiative for fibrosis treatment (GIFT).

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterised by increased scarring of lung tissue. Despite the recent introduction of novel drugs that slow disease progression, IPF remains a deadly disease, and the benefits of these new drugs differ markedly between patients. Human diseases arise due to alterations in an almost limitless network of interconnected genes, proteins, metabolites, cells and tissues, in direct relationship with a continuously changing macro- or microenvironment. Systems biology is a novel research strategy that seeks to understand the structure and behaviour of the so-called "emergent properties" of complex systems, such as those involved in disease pathogenesis, which are most often overlooked when just one element of disease pathogenesis is observed in isolation. This article summarises the debate that took place during a European Respiratory Society research seminar in Barcelona, Spain on December 15-16, 2016, which focused on how systems biology could generate new data by integrating the different IPF pathogenic levels of complexity. The main conclusion of the seminar was to create a global initiative to improve IPF outcomes by integrating cutting-edge international research that leverages systems biology to develop a precision medicine approach to tackle this devastating disease